2,5-Dkg permeases

ABSTRACT

The invention provides isolated nucleic acid molecules encoding polypeptides having 2,5-DKG permease activity, and oligonucleotides therefrom. The isolated nucleic acid molecules can be expressed in appropriate bacterial cells to enhance the production of 2-KLG, which can subsequently be converted to ascorbic acid. Further provided are isolated polypeptides having 2,5-DKG permease acitivity, immunogenic peptides therefrom, and antibodies specific therefor. The invention also provides methods of identifying novel 2,5-DKG permeases.

[0001] This invention was made in part with U.S. Government supportunder Cooperative Agreement 70NANB5H1138 and ATP NIST projectIdentification Number 1995-05-0007E. The U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to microbial transporterproteins and, more specifically, to novel 2,5-diketo-D-gluconic acid(2,5-DKG) permeases.

[0004] 2. Background Information

[0005] Adequate intake of ascorbic acid, or vitamin C, is recognized asan important factor in maintaining health. To ensure adequate intake ofascorbic acid, the chemical is now added to many foods, drinks andcosmetic products, and is also sold as a direct vitamin supplement. Tomeet the commercial demand for ascorbic acid, there is a need to developmore efficient processes for its production.

[0006] Although there are a number of alternative methods of producingascorbic acid, one of the least expensive and most ecologically soundmethods is biofermentation. Bacterial strains have now been engineeredto express all of the enzymes required for the stepwise conversion of aninexpensive sugar source, such as D-glucose, to a stable precursor ofascorbic acid, 2-keto-L-gulonic acid (2-KLG) (see U.S. Pat. No.5,032,514 and references therein). 2-KLG can be readily converted toascorbic acid by chemical or enzymatic procedures.

[0007]FIG. 2 shows schematically the enzymatic reactions that take placein the bioconversion of D-glucose to 2-KLG. As shown in FIG. 2, theenzymatic reactions that lead from D-glucose, to D-gluconic acid, to2-keto-D-gluconic acid (2-KDG), to 2,5-diketo-D-gluconic acid (2,5-DKG),take place at the surface of the bacterial cell. 2,5-DKG must then enterthe cell in order for its enzymatic conversion to 2-KLG.

[0008] Much effort has been expended in increasing the efficiency of theenzymatic reactions involved in 2-KLG production. For example, U.S. Pat.No. 5,032,514 describes methods for increasing 2-KLG production byreducing metabolic diversion of 2,5-DKG to products other than 2-KLG.

[0009] Increasing uptake of 2,5-DKG by a bacterial strain suitable forbiofermentation could be advantageous in increasing 2-KLG production.Expressing additional copies of an endogenous 2,5-DKG permease, orexpressing an exogenous 2,5-DKG permease with superior properties, couldincrease uptake of 2,5-DKG. However, to date, no 2,5-DKG permease hasbeen identified or characterized that could be used in this manner.

[0010] Therefore, there exists a need to identify and characterizenucleic acid molecules encoding 2,5-DKG permeases, so that permeaseswith advantageous properties can be used in the commercial production ofascorbic acid and in other important applications. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0011] The invention provides an isolated nucleic acid molecule encodinga polypeptide which has 2,5-DKG permease activity. In one embodiment,the isolated nucleic acid molecule contains a nucleotide sequence havingat least 40% identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7, 9 and 11. In another embodiment,the isolated nucleic acid molecule contains a nucleotide sequence whichencodes a polypeptide having at least 40% identity to an amino acidsequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8,10 and 12.

[0012] Also provided are vectors and cells containing isolated nucleicacid molecules encoding polypeptides having 2,5-KDG permease activity.In one embodiment, the cells are bacterial cells selected from thegenera Pantoea and Klebsiella.

[0013] The invention also provides methods of identifying and isolatingnucleic acid molecules encoding polypeptides which have 2,5-DKG permeaseactivity. Also provided are methods of enhancing 2-KLG production, byexpressing the nucleic acid molecules of the invention in suitablebacterial cells.

[0014] Further provided are isolated polypeptides having 2,5-DKGpermease activity, and immunogenic peptides therefrom. The inventionalso provides antibodies specific for such polypeptides and peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIGS. 1A and 1B shows an alignment of the amino acid sequences ofthe 2,5-DKG permeases designated YiaX2 (SEQ ID NO: 12); PE1 (SEQ ID NO:2); PE6 (SEQ ID NO: 4); prmA (SEQ ID NO: 8); prmB (SEQ ID NO: 10) andPK1 (SEQ ID NO: 6).

[0016]FIG. 2 shows the biosynthetic pathway from glucose to 2-KLG in abacterial strain suitable for biofermentation.

[0017]FIG. 3 shows the metabolic selection strategy used to identifynovel 2,5-DKG permeases.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The invention provides novel nucleic acid molecules encodingpolypeptides having 2,5-DKG permease activity, and related products andmethods. The molecules of the invention can advantageously be used toincrease the efficiency of 2-KLG bioproduction, and thus to lower thecost of commercial ascorbic acid production.

[0019] Naturally occurring 2,5-DKG permeases are polypeptides localizedto the cytoplasmic membrane of microorganisms, which are predicted,using commercially available topology prediction programs, to containabout 10 to 12 transmembrane domains. Each transmembrane spanningsegment is about 20 amino acids in length, with the intracellular andextracellular loops ranging from about 2 to about 83 amino acids inlength. Generally, the loop between the fifth and sixth transmembranedomain spanning segments is larger than the other loops.

[0020] Naturally occurring 2,5-DKG permeases are typically about 350-550amino acids in length, such as about 400-450 amino acids in length, andparticularly about 425-440 amino acids in length.

[0021] The nucleotide sequences encoding six exemplary 2,5-DKG permeasesare set forth as follows, with the designation and organismal source ofthe molecule indicated in parentheses: SEQ ID NO: 1 (PE1 from anenvironmental source); SEQ ID NO: 3 (PE6 from an environmental source);SEQ ID NO: 5 (PK1 from Klebsiella oxytoca); SEQ ID NO: 7 (prmA fromPantoea citrea); SEQ ID NO: 9 (prmB from Pantoea citrea); and SEQ ID NO:11 (YiaX2 from Klebsiella oxytoca). The corresponding encoded 2,5-DKGpermease amino acid sequences are set forth as SEQ ID NO: 2 (PE1); SEQID NO: 4 (PE6); SEQ ID NO: 6 (PK1) ; SEQ ID NO: 8 (prmA); SEQ ID NO: 10(prmB); and SEQ ID NO: 12 (YiaX2).

[0022] 2,5-DKG permeases from different microorganisms exhibit extensiveamino acid sequence relatedness over their entire length, as isevidenced by the six-way sequence alignment shown in FIG. 1. The overallidentity of the six permeases shown in FIG. 1 is about 17%, and theoverall similarity, taking into account conservative substitutions, isabout 43%.

[0023] Based on their predicted topological and sequence similarity,2,5-DKG permeases disclosed herein can be further subdivided into twostructural families. The three 2,5-DKG permeases designated YiaX2, PE6and PrmA are representative of one family of permeases, sharing about50% overall identity in a three-way amino acid sequence alignment. Thethree 2,5-DKG permeases designated PK1, PE1 and PrmB are representativeof a second family of related permeases, sharing about 60% overallidentity in a three-way amino acid sequence alignment.

[0024] Naturally occurring 2,5-DKG permeases also exhibit 2,5-DKGpermease activity. The term “2,5-DKG permease activity,” as used herein,refers to the ability of the polypeptide, when expressed in its nativeorientation at the cell membrane, to transport 2,5-DKG across thecytoplasmic membrane, in comparison with an unrelated controlpolypeptide. Such transport can be either unidirectional orbidirectional.

[0025] 2,5-DKG permease activity can be determined by a variety ofmethods. For example, 2,5-DKG permease activity can be determined usinga metabolic selection assay, as described further in the Example, below.Briefly, a bacterial cell either naturally deficient in 2,5-DKG permeaseactivity, or made deficient in 2,5-DKG permease activity, is identifiedor produced. As described in the Example, bacterial cells can be madedeficient in endogenous 2,5-DKG permease activity by preparing adeletion mutant of one or more endogenous 2,5-DKG permease genes, usingthe polymerase chain reaction, following methods known in the art. Theterm “deficient,” as used in relation to a cell deficient in 2,5-DKGpermease activity, is intended to refer to endogenous 2,5-DKG permeaseactivity that is comparable to, or less than, the endogenous permeaseactivity of a K. oxytoca strain deleted in the yiaX2 gene, such as thestrain K. oxytoca ΔyiaX2 [tkr idnO], as assessed either by a growthassay or by a 2,5-DKG uptake assay.

[0026] A cell useful in a metabolic selection assay to determine 2,5-DKGpermease activity of an expressed polypeptide can further naturally becapable of converting intracellular 2,5-DKG to carbon and energy, ormade capable of such conversion by recombinant expression of appropriatemetabolic enzymes. As described in the Example, a combination of nucleicacid molecules encoding a 2-keto-reductase (tkr) and a 5-keto-reductase(idnO), from any bacterial species, can be expressed in the cell, whichtogether provide the cell with the ability to catalyze the reduction of2,5-DKG to gluconic acid. Gluconic acid can then be used by the cell asa carbon and energy source that supports cell growth.

[0027] An exemplary bacterial cell suitable for metabolic assays todetermine 2,5-DKG permease activity is the strain K. oxytoca ΔyiaX2 [tkridnO] shown in FIG. 3 and described in the Example, below. This strainhas a deleted yiaX2 2,5-DKG permease gene, and also recombinantlyexpresses the tkr/idnD/idnO operon set forth as SEQ ID NO: 13 on a highcopy number plasmid. Within SEQ ID NO: 13, nucleotides 292-1236 encode a2-keto-reductase (tkr) (SEQ ID NO: 14); nucleotides 1252-2280 encode anidonic acid dehydrogenase (idnD) (SEQ ID NO: 15); and nucleotides2293-3045 encode a 5-keto-reductase (idnO) (SEQ ID NO: 16).Alternatively, nucleic acid molecules encoding polypeptides whichcontain modifications from the amino acid sequences designated SEQ IDNO: 14 or 16, but which retain 2-keto-reductase activity or5-keto-reductase activity, respectively, can be used in metabolicassays. Exemplary amino acid sequences have at least 60%, such as atleast 70%, preferably 80%, 90%, 95% or greater identity to SEQ ID NOS:14 or 16, respectively.

[0028] The ability of such a bacterial cell to grow on medium containing2,5-DKG as the sole carbon source, upon expression of a candidate2,5-DKG permease, is a measure of the ability of the expressed permeaseto transport 2,5-DKG into the cell, and is thus a measure of its 2,5-DKGpermease activity. Each of SEQ ID NOS: 2, 4, 6, 8, 10 and 12 wasdemonstrated to have 2,5-DKG permease activity, as evidenced by theability of K. oxytoca ΔyiaX2 [tkr idnO] expressing each permease to growon 2,5-DKG as the sole carbon source.

[0029] Likewise, 2,5-DKG permease activity can be determined bymeasuring uptake of labeled or unlabeled 2,5-DKG. For example, 2,5-DKGcan be detectably labeled, such as with a fluorescent or radioactivetag. The ability of a cell or membrane vesicle expressing a 2,5-DKGpermease to take up the detectable label when provided with detectablylabeled 2,5-DKG, can be determined using detection assays specific forthe particular label, which are well known in the art. Likewise, uptakeof unlabeled 2,5-DKG can be measured by HPLC or other sensitivedetection assay known in the art. Uptake of 2,5-DKG is thus a measure ofpermease activity. Each of SEQ ID NOS: 2, 4, 6, 8, 10 and 12 exhibits2,5-DKG permease activity as determined by assay of uptake ofradiolabeled 2,5-DKG by bacterial cells expressing the recombinantpermeases.

[0030] Additionally, 2,5-DKG permease activity can be measured in anycell in which 2,5-DKG can be converted to a product, by measuringproduction of the product in the presence of extracellular 2,5-DKG. Forexample, in a cell naturally expressing, or recombinantly expressing, a2,5-DKG reductase, intracellular 2,5-DKG is converted to 2-KLG. Theability of the bacterial cell to produce 2-KLG when provided withextracellular 2,5-DKG, upon expression of a 2,5-DKG permease, is ameasure of the ability of the expressed permease to transport 2,5-DKGinto the cell, and is thus a measure of its 2,5-DKG permease activity.Intracellular 2-KLG can be detected, for example, using HPLC or othersensitive detection methods known in the art. Other metabolic productsof 2,5-DKG can also be detected, by similar methods.

[0031] It will be appreciated that a variety of alternative assays canbe used to determine 2,5-DKG permease activity. For instance, the changein pH across a cell or vesicle membrane as 2,5-DKG, an acid, istransported across the membrane can be detected. Similarly, a decreaseover time in extracellular 2,5-DKG can be determined.

[0032] Accordingly, using any of the activity assays described herein,those skilled in the art can distinguish between a polypeptide having2,5-DKG permease activity, and a polypeptide not having such activity.

[0033] A 2,5-DKG permease of the invention can selectively transport2,5-DKG. As used herein in relation to transport activity, the term“selective” refers to preferential transport of 2,5-DKG rather than2-KLG into or out of the cell. A permease that selectively transports2,5-DKG will transport 2,5-DKG at least 2-fold, such as at least 5-fold,including greater than 10-fold more efficiently than it transports2-KLG. A permease that selectively transports 2,5-DKG is particularlyadvantageous in applications where it is desirable to increaseintracellular production of 2-KLG, such as in the commercial productionof ascorbic acid. In particular, employing a permease that selectivelytransports 2,5-DKG prevents intracellular 2-KLG from competing withextracellular 2,5-DKG for permease-mediated transport through themembrane, and increases the overall efficiency of intracellular 2-KLGproduction.

[0034] It will be appreciated that the assays described above fordetermining 2,5-DKG permease activity can be modified to simultaneously,or separately, determine 2-KLG permease activity. For example, ametabolic assay can be designed in which a bacterial cell can converteither intracellular 2,5-DKG or 2-KLG to carbon and energy. In such acell, the relative ability of the cell to grow on 2,5-DKG as the solecarbon source, compared with its ability to grow on 2-KLG as the solecarbon source, is a measure of the ability of the expressed permease toselectively transport 2,5-DKG. Using such an assay, it was determinedthat the 2,5-DKG permeases designated YiaX2, PE1, PE6, prmA and prmB arenon-selective for 2,5-DKG, as they also efficiently catalyze thetransport of 2-KLG, as K. oxytoca ΔyiaX2 [tkr idnO] cells expressingsuch permeases grow well on either 2,5-DKG or 2-KLG. In contrast, PK1selectively transports 2,5-DKG, and K. oxytoca ΔyiaX2 [tkr idnO] cellsexpressing PK1 (SEQ ID NO: 6) grow on 2,5-DKG but not on 2-KLG as thesole carbon source.

[0035] The invention provides an isolated nucleic acid molecule encodinga polypeptide which has 2,5-DKG permease activity. The invention nucleicacid molecules of the invention are suitable for a variety of commercialand research applications. For example, one or more of the inventionnucleic acid molecules can be expressed in bacterial cells in order toenhance the rate of uptake of 2,5-DKG by the cells. Enhancing uptake of2,5-DKG has a variety of applications, such as in commercial productionof 2,5-DKG itself, or commercial production of any metabolic product of2,5-DKG. For example, 2,5-DKG uptake is a rate limiting step in thebiosynthesis of 2-KLG, which is a stable intermediate in the synthesisof ascorbic acid. 2-KLG can thus be obtained from bacterial cellsexpressing 2,5-DKG permeases, and converted to ascorbic acid.

[0036] Additionally, the invention nucleic acid molecules can be used asprobes or primers to identify and isolate 2,5-DKG permease homologs fromadditional species, or as templates for the production of mutantpermeases, using methods known in the art and described further below.Such permeases can have advantageous properties compared with the2,5-DKG permeases disclosed herein as SEQ ID NOS: 2, 4, 6, 8, 10 and 12,such as greater enzymatic activity or greater 2,5-DKG selectivity.

[0037] In one embodiment, an isolated nucleic acid molecule of theinvention is not completely contained within the nucleotide sequencedesignated SEQ ID NO: 19 of WO 00/22170, which is the K. oxytoca yiaoperon. In another embodiment, the isolated nucleic acid molecule of theinvention is not completely contained within the nucleotide sequenceherein designated SEQ ID NO: 11. In another embodiment, the encodedpolypeptide is not completely contained within the amino acid sequenceherein designated SEQ ID NO: 12.

[0038] The term “isolated,” as used herein, is intended to mean that themolecule is altered, by the hand of man, from how it is found in itsnatural environment. For example, an isolated nucleic acid molecule canbe a molecule operatively linked to an exogenous nucleic acid sequence.An isolated nucleic acid molecule can also be a molecule removed fromsome or all of its normal flanking nucleic acid sequences, such asremoved from one or more other genes within the operon in which thenucleic acid molecule is normally found.

[0039] Specifically with respect to an isolated nucleic acid moleculecontaining the nucleotide sequence designated SEQ ID NO: 11, or encodingthe yiaX2 polypeptide designated SEQ ID NO: 12, the term “isolated” isintended to mean that the nucleic acid molecule does not contain any ofthe flanking open reading frames (orfs) present in the K. oxytoca yiaoperon, such as the orfs designated lyxK and orf1, described in WO00/22170.

[0040] An isolated molecule can alternatively, or additionally, be a“substantially pure” molecule, in that the molecule is at least 60%,70%, 80%, 90 or 95% free from cellular components with which it isnaturally associated. An isolated nucleic acid molecule can be in anyform, such as in a buffered solution, a suspension, a heterologous cell,a lyophilized powder, or attached to a solid support.

[0041] The term “nucleic acid molecule” as used herein refers to apolynucleotide of natural or synthetic origin. A nucleic acid moleculecan be single- or double-stranded genomic DNA, cDNA or RNA, andrepresent either the sense or antisense strand or both. A nucleic acidmolecule can thus correspond to the recited sequence, to its complement,or both.

[0042] The term “nucleic acid molecule” is intended to include nucleicacid molecules that contain one or more non-natural nucleotides, such asnucleotides having modifications to the base, the sugar, or thephosphate portion, or having one or more non-natural linkages, such asphosphothioate linkages. Such modifications can be advantageous inincreasing the stability of the nucleic acid molecule, particularly whenused in hybridization applications.

[0043] Furthermore, the term “nucleic acid molecule” is intended toinclude nucleic acid molecules modified to contain a detectable moiety,such as a radiolabel, a fluorochrome, a ferromagnetic substance, aluminescent tag or a detectable binding agent such as biotin. Nucleicacid molecules containing such moieties are useful as probes fordetecting the presence or expression of a 2,5-DKG permease nucleic acidmolecule.

[0044] In one embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity contains a nucleotidesequence comprising nucleotides 1-20, 1-100, 101-120, 101-200, 201-220,201-300, 301-320, 301-400, 401-420, 401-500, 501-520, 501-600, 601-620,601-700, 701-720, 701-800, 801-820, 801-900, 901-920, 901-1000,1001-1020, 1001-1100, 1101-1120, 1100-1200, 1201-1220, 1201-1300,1301-1320, 1301-1400, 1401-1420 or 1401-1500 of any of SEQ ID NOS: 1, 3,5, 7, 9 or 11.

[0045] In another embodiment, the isolated nucleic acid moleculeencoding a polypeptide which has 2,5-DKG permease activity encodes anamino acid sequence comprising amino acids 1-10, 1-50, 51-60, 51-100,101-110, 101-150, 151-160, 151-200, 201-210, 201-250, 251-260, 251-300,301-310, 301-350, 351-361, 351-400, 401-410 or 401-439 of any of SEQ IDNOS: 2, 4, 6, 8, 10 or 12.

[0046] In one embodiment, the isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity contains a nucleotidesequence having at least 35% identity to any of the 2,5-DKG permeasenucleic acid molecules designated SEQ ID NOS: 1, 3, 5, 7, 9 or 11.Preferably, such a molecule will have at least 40% identity to any ofthese recited SEQ ID NOS, such as at least 45%, 50%, 60%, 70% or 80%identity, including at least 90%, 95%, 98%, 99% or greater identity toSEQ ID NOS: 1, 3, 5, 7, 9 or 11.

[0047] In another embodiment, the isolated nucleic acid moleculeencoding a polypeptide which has 2,5-DKG permease activity contains anucleotide sequence which encodes a polypeptide having at least 35%identity to any of the 2,5-DKG permease polypeptides designated SEQ IDNOS: 2, 4, 6, 8, 10 or 12. Preferably, the encoded polypeptide will haveat least 40% identity to any of these recited SEQ ID NOS, such as atleast 45%, 50%, 60%, 70%, 80% identity, including at least 90%, 95%,98%, 99% or greater identity to SEQ ID NOS: 2, 4, 6, 8, 10 or 12.

[0048] The term “percent identity” with respect to a nucleic acidmolecule or polypeptide of the invention is intended to refer to thenumber of identical nucleotide or amino acid residues between thealigned portions of two sequences, expressed as a percent of the totalnumber of aligned residues, as determined by comparing the entiresequences using a CLUSTAL V computer alignment and default parameters.CLUSTAL V alignments are described in Higgens, Methods Mol. Biol.25:307-318 (1994), and an exemplary CLUSTAL V alignment of 2,5-DKGpermease amino acid sequences is presented in FIG. 1.

[0049] Due to the degeneracy of the genetic code, the nucleotidesequence of a native nucleic acid molecule can be modified and stillencode an identical or substantially similar polypeptide. Thus,degenerate variants of SEQ ID NOS: 1, 3, 5, 7, 9 or 11 are exemplaryinvention nucleic acid molecules encoding polypeptides having 2,5-DKGpermease activity.

[0050] Additionally, nucleic acid molecules encoding 2,5-DKG permeasesfrom other species of microorganisms are exemplary invention nucleicacid molecules. The six permeases designated YiaX2, PE1, PE6, prmA, prmBand PK1, which were isolated from at least three, and likely four,different species of microorganisms, share substantial nucleotidesequence identity. For example, the two most similar of the disclosed2,5-DKG permease nucleotide sequences, SEQ ID NO: 5 (PK1) and SEQ ID NO:1 (PE1), share 86% identity across their length. The two most dissimilarof the disclosed 2,5-DKG permease nucleotide sequences, SEQ ID NO: 7(prmA) and SEQ ID NO: 9 (prmB), share 51% identity across their length.In contrast, a search of GenBank reveals no other nucleotide sequences,including sequences which encode transporter proteins and othertransmembrane proteins, that exhibit significant identity or similarityto any of the disclosed 2,5-DKG permease nucleotide sequences over theentire length of their sequences.

[0051] The six permeases disclosed herein also share substantial aminoacid sequence identity over their entire length, as describedpreviously. For example, PK1 from Klebsiella oxytoca (SEQ ID NO: 6), andPE1, from an environmental source (SEQ ID NO: 2), are 93% identical atthe amino acid level. The amino acid sequence in the GenBank databasemost closely related to a 2,5-DKG permease, which is a putative tartratetransporter from Agrobacterium vitis (GenBank Accession U32375 orU25634) is 33% identical to SEQ ID NO: 12 (YiaX2), and shares lessidentity with the other disclosed 2,5-DKG permeases. Other sequenceswith some degree of identity in the GenBank database to the disclosed2,5-DKG permease include membrane transporter proteins from a variety ofspecies, including phthalate transporter proteins from B. cepacia(AF152094) and P. putida (D13229); hydroxyphenylacetate transportersfrom S. dublin (AF144422) and E. coli (Z37980); and probable transporterproteins from S. coelicolor (AL136503 and AL132991) each of which hasabout 27% or less identity at the amino acid level to the recited SEQ IDNOS.

[0052] In view of the high degree of identity between different 2,5-DKGpermease nucleic acid molecules and encoded polypeptides within a singlespecies and between different microbial species, additional 2,5-DKGpermeases from other species can be readily identified and tested. Thus,nucleic acid molecules of the invention include nucleic acid moleculesthat encode polypeptides having 2,5-DKG permease activity from anymicrobial species. Microorganisms that contain 2,5-DKG permeases can berecognized by their ability to actively transport 2,5-DKG, such thatthey can grow on 2,5-DKG as the sole carbon source, or incorporate2,5-DKG in an uptake assay. Such microorganisms can include, forexample, bacteria, including Archaebacteria, gram positive and gramnegative bacteria; yeast; and fungi.

[0053] Exemplary bacteria which contain 2,5-DKG permeases includeProteobacteria, and more specifically Enterobacteria and Pseudomonads(e.g. P. aeruginosa), as described in the Example. ExemplaryEnterobacteria include species from the genera Klebsiella (e.g. K.oxytoca, from which SEQ ID NOS: 5 and 11 were obtained) and Pantoea(e.g. P. citrea, from which SEQ ID NOS: 7 and 9 were obtained, and P.agglomerans). Sources of such microorganisms include publicrepositories, such as the American Type Culture Collection (ATCC), andcommercial sources. It will be appreciated that the taxonomy andnomenclature of bacterial genera are such that the same or similarstrains are sometimes reported in the literature as having differentnames. For example, Klebsiella oxytoca (e.g. ATCC 13182) hasalternatively been described as Aerobacter aerogenes, Klebsiellaaerogenes and Klebsiella pneumoniae. Likewise, Pantoea agglomerans (e.g.ATCC 21998) has alternatively been described as Erwinia herbicola andAcetomonas albosesamae. The terms “Klebsiella” and “Pantoea,” as usedherein, are intended to refer to the genera of the strains deposited asATCC 13182 and 21998, respectively.

[0054] Additionally, microorganisms from which 2,5-DKG permease nucleicacid molecules can be obtained are microorganisms present inenvironmental samples. For example, the 2,5-DKG permease nucleic acidmolecules designated SEQ ID NOS: 1 and 3 were obtained fromenvironmental samples. As used herein, the term “environmental sample”refers to a sample obtained from natural or man-made environments, whichgenerally contains a mixture of microorganisms.

[0055] Exemplary environmental samples are samples of soil, sand,freshwater or freshwater sediments, marine water or marine watersediments, industrial effluents, hot springs, thermal vents, and thelike. Within an environmental sample there are likely to bemicroorganisms that are unidentified, and also microorganisms that areuncultivable. Isolation of invention 2,5-DKG permease molecules of theinvention from microorganisms present in environmental samples does notrequire either identification or culturing of the microorganism.

[0056] Furthermore, nucleic acid molecules of the invention includenucleic acid molecules encoding amino acid sequences that are modifiedby one or more amino acid additions, deletions or substitutions withrespect to the native sequence of SEQ ID NOS: 2, 4, 6, 8, 10 or 12. Suchmodifications can be advantageous, for example, in enhancing thestability, expression level, enzymatic activity, or 2,5-DKG selectivityof the permease. If desired, such modifications can be randomlygenerated, such as by chemical mutagenesis, or directed, such as bysite-directed mutagenesis of a native permease sequence, using methodswell known in the art.

[0057] An amino acid sequence that is modified from a native permeaseamino acid sequence can include one or more conservative amino acidsubstitutions, such as substitution of an apolar amino acid with anotherapolar amino acid (such as replacement of leucine with an isoleucine,valine, alanine, proline, tryptophan, phenylalanine or methionine);substitution of a charged amino acid with a similarly charged amino acid(such as replacement of a glutamic acid with an aspartic acid, orreplacement of an arginine with a lysine or histidine); or substitutionof an uncharged polar amino acid with another uncharged polar amino acid(such as replacement of a serine with a glycine, threonine, tyrosine,cysteine, asparagine or glutamine). A modified amino acid sequence canalso include one or more nonconservative substitutions without adverselyaffecting the desired biological activity.

[0058] Computer programs known in the art can provide guidance indetermining which amino acid residues can be substituted withoutabolishing the enzymatic activity of a 2,5-DKG permease (see, forexample, Eroshkin et al., Comput. Appl. Biosci. 9:491-497 (1993)).

[0059] Additionally, guidance in modifying amino acid sequences whileretaining or enhancing functional activity is provided by aligninghomologous 2,5-DKG permease polypeptides from various species (see FIG.1). It is well known in the art that evolutionarily conserved amino acidresidues and domains are more likely to be important for maintainingbiological activity than less well-conserved residues and domains. Thus,it would be expected that substituting a residue which is highlyconserved among the six 2,5-DKG permeases shown in FIG. 1 (or among themembers of the two structural families of permeases, defined as SEQ IDNOS: 2, 6 and 10, and SEQ ID NOS: 4, 8 and 12) with a non-conservedresidue may be deleterious, whereas making the same substitution at aresidue which varies widely among the different permeases would likelynot have a significant effect on biological activity.

[0060] A comparison of the amino acid sequences of PE1 (SEQ ID NO: 2),which transports both 2,5-DKG and 2-KLG, and PK1 (SEQ ID NO: 6), whichselectively transports 2,5-DKG, indicates that the regions responsiblefor 2,5-DKG selectivity must reside in the 7% of amino acids whichdiffer between these two sequences. Therefore, modifying all or some ofthese differing residues in a 2,5-DKG permease to those found in the PK1sequence would be expected to increase 2,5-DKG selectivity of thepermease.

[0061] Alignment of the six 2,5-DKG permeases described herein alsoprovides guidance as to regions where additions and deletions are likelyto be tolerated. For example the N and C termini, and the region aroundamino acids 225-250 (based on the numbering of SEQ ID NO: 12 (yiaX2))appear to be regions that are relatively tolerant of amino acidinsertions and deletions, as evidenced by gaps in the sequencealignment. Modified 2,5-DKG permeases can thus include “tag” sequencesat such sites, such as epitope tags, histidine tags,glutathione-S-transferase (GST) and the like, or sorting sequences. Suchadditional sequences can be used, for example, to facilitatepurification or characterization of a recombinant 2,5-DKG permease.

[0062] It will be appreciated that confirmation that any particularnucleic acid molecule is a nucleic acid molecule of the invention can beobtained by determining the 2,5-DKG permease activity of the encodedpolypeptide, using one or more of the functional assays describedherein.

[0063] The invention further provides an isolated nucleic acid moleculeencoding a polypeptide which has 2,5-DKG permease activity, wherein thenucleic acid molecule is operatively linked to a promoter of geneexpression. The term “operatively linked,” as used herein, is intendedto mean that the nucleic acid molecule is positioned with respect toeither the endogenous promoter, or a heterologous promoter, in such amanner that the promoter will direct the transcription of RNA using thenucleic acid molecule as a template.

[0064] Methods for operatively linking a nucleic acid to a desiredpromoter are well known in the art and include, for example, cloning thenucleic acid into a vector containing the desired promoter, or appendingthe promoter to a nucleic acid sequence using PCR. A nucleic acidmolecule operatively linked to a promoter of RNA transcription can beused to express 2,5-DKG transcripts and polypeptides in a desired hostcell or in vitro transcription-translation system.

[0065] The choice of promoter to operatively link to an inventionnucleic acid molecule will depend on the intended application, and canbe determined by those skilled in the art. For example, if a particulargene product may be detrimental to a particular host cell, it may bedesirable to link the invention nucleic acid molecule to a regulatedpromoter, such that gene expression can be turned on or off. Anexemplary inducible promoter known in the art is the lacPOpromoter/operator, which is repressed by the lacI^(q) gene productprovided by certain host cells, and induced in the presence of 0.01 to 1mM IPTG (see Example, below). For other applications, weak or strongconstitutive promoters may be preferred.

[0066] The invention further provides a vector containing an isolatednucleic acid molecule encoding a polypeptide which has 2,5-DKG permeaseactivity. The vectors of the invention will generally contain elementssuch as a bacterial origin of replication, one or more selectablemarkers, and one ore more multiple cloning sites. The choice ofparticular elements to include in a vector will depend on factors suchas the intended host cell or cells; whether expression of the insertedsequence is desired; the desired copy number of the vector; the desiredselection system, and the like. The factors involved in ensuringcompatibility between a host and a vector for different applications arewell known in the art.

[0067] In applications in which the vectors will be used for recombinantexpression of the encoded polypeptide, the isolated nucleic acidmolecules will generally be operatively linked to a promoter of geneexpression, as described above, which may be present in the vector or inthe inserted nucleic acid molecule. In cloning and subcloningapplications, however, promoter elements need not be present.

[0068] An exemplary vector suitable for both cloning applications andfor expressing 2,5-DKG permeases in different bacterial species is thelow copy number plasmid pCL1920 described by Lerner et al., NucleicAcids Res. 18:4621 (1994), which contains a spectinomycin resistancegene (see Example, below).

[0069] Also provided are cells containing an isolated nucleic acidmolecule encoding a polypeptide which has 2,5-DKG permease activity. Theisolated nucleic acid molecule will generally be contained within avector compatible with replication in the particular host cell. However,for certain applications, incorporation of the nucleic acid moleculeinto the bacterial genome will be preferable.

[0070] The cells of the invention can be any cells in which a 2,5-DKGpermease will be expressed and folded into an active conformation.Guidance in choosing appropriate host cells is provided by identifyingcell types which express other functional 10 to 12 transmembranetransporter proteins. For example, 10 to 12 transmembrane transporterproteins are found in a variety of bacterial species, as well as inyeast (e.g. S. pombe), Arabidopsis, and Drosophila. Therefore, dependingon the particular application for the host cell, a host cell of theinvention can be a bacterial cell, yeast, Arabidopsis or Drosophilacell.

[0071] In a preferred embodiment, the cell is a bacterial cell. Thechoice of bacterial cell will depend on the intended application. Forexample, for routine subcloning applications, the cell can be anyconvenient laboratory strain of bacteria, such as E. coli, which can betransformed with the isolated nucleic acid molecules and vectors of theinvention by methods well known in the art.

[0072] For assessment of encoded 2,5-DKG permease activity, the cell canbe a bacterial strain suitable for metabolic assays, such as a strainwhich endogenously expresses, or which is engineered to express, enzymesthat catalyze the conversion of 2,5-DKG to essential products. Anexemplary strain suitable for metabolic assays is the K. oxytoca ΔyiaX2[tkr idnO] strain designated MGK002[pDF33] described further in theExample, below, which provides for the conversion of intracellular2,5-DKG to gluconic acid, which can be used as a carbon and energysource.

[0073] For use in the commercial bioproduction of 2,5-DKG metabolites,the cell can be a bacterial strain which endogenously expresses, orwhich is engineered to express, a 2,5-DKG reductase. As described inU.S. Pat. No. 5,032,514, 2,5-DKG reductases are found in generaincluding Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus,Pseudomonas, Bacillus, Citrobacter and Corynebacterium. Therefore, acell of the invention can be a bacterial cell of any of these genera, ora bacterial cell engineered to express a 2,5-DKG reductase of any ofthese genera.

[0074] A cell able to produce 2,5-DKG metabolites will preferably alsobe able to catalyze the extracellular production of 2,5-DKG from aninexpensive carbon source, such as glucose. An exemplary pathway fromD-glucose to 2,5-DKG involves the enzymatic conversion of D-glucose toD-gluconic acid (catalyzed by D-glucose dehydrogenase), from D-gluconicacid to 2-Keto-D-gluconic acid (catalyzed by D-gluconate dehydrogenase),and from 2-Keto-D-gluconic acid to 2,5-DKG (catalyzed by2-Keto-D-gluconic acid dehydrogenase), as is shown in FIG. 2. Thesesteps can be carried out by organisms of several genera, includingGluconobacter, Acetobacter and Erwinia (also called Pantoea).

[0075] A bacterial cell useful for the production of 2-KLG fromD-glucose is the Pantoea aggolmerans (also referred to as Erwiniaherbicola or Acetomonas albosesamae) strain described in U.S. Pat. No.5,032,514, designated ATCC 21998 ptrp 1-35 tkrAΔ3, or a derivative ofthis strain with improved properties. Contemplated improvements to thisstrain, which can be produced by genetic engineering, include deletionof enzymes that divert glucose to metabolites other than 2-KLG, suchthat yield of 2-KLG is increased. Other contemplated improvements tothis strain include mutations that provide for improved recovery andpurification of 2-KLG.

[0076] The Pantoea strain described in U.S. Pat. No. 5,032,514recombinantly expresses a 2,5-DKG reductase from Corynebacterium(described in U.S. Pat. No. 4,757,012). The strain further contains amutation that results in a non-functional tkrA gene and is thusdeficient in 2-keto reductase activity. Mutation of the tkrA gene isadvantageous in reducing metabolic diversion of 2-KLG to L-idonic acid,and metabolic diversion of 2,5-DKG to 5-keto-D-gluconate from 2-KLG.

[0077] Expression of one or more 2,5-DKG permeases of the invention insuch cells significantly increases overall production of 2-KLG fromD-glucose, which lowers the cost of commercial production of ascorbicacid.

[0078] The cells of the invention can contain one, two or more isolatednucleic acid molecules of the invention that encode polypeptides having2,5-DKG permease activity. For example, the cell can contain an isolatednucleic acid molecule encoding at least one polypeptide having at least80% identity to any of SEQ ID NOS: 2, 4, 6, 8, 10 or 12, and optionallywill contain two or more such nucleic acid molecules, in anycombination. Preferably, at least one such encoded polypeptideselectively transports 2,5-DKG.

[0079] In a preferred embodiment, a bacterial cell of the inventionsuitable for bioproduction of 2-KLG contains an isolated nucleic acidmolecule encoding a polypeptide having at least 95% identity to the2,5-DKG selective permease designated SEQ ID NO: 8 (prmA); andoptionally further containing at least one isolated nucleic acidmolecule encoding a polypeptide having at least 95% identity to a2,5-DKG permease selected from the group consisting of SEQ ID NO: 4(PE6), SEQ ID NO: 10 (prmB) and SEQ ID NO: 6 (PK1).

[0080] The invention also provides a method of enhancing production of2-KLG. The method consists of culturing a bacterial cell, wherein thecell contains an isolated nucleic acid molecule encoding a polypeptidewhich has 2,5-DKG permease activity, under conditions wherein theencoded 2,5-DKG permease is expressed and intracellular 2,5-DKG isconverted to 2-KLG. Cells suitable for this purpose, such as the Pantoeastrain described in U.S. Pat. No. 5,032,514, have been described above.Optionally, the 2-KLG so produced can be chemically or enzymaticallyconverted to a desired product such as ascorbic acid, following methodsknown in the art.

[0081] The invention further provides isolated oligonucleotide moleculesthat contain at least 17 contiguous nucleotides from any of thenucleotide sequences referenced as SEQ ID NOS: 1, 3, 5, 7, 9 or 11. Asused herein, the term “oligonucleotide” refers to a nucleic acidmolecule that contains at least 17 contiguous nucleotides from thereference sequence and which may, but need not, encode a functionalprotein. Thus, an oligonucleotide of the invention can contain at least18, 19, 20, 22 or 25 contiguous nucleotides, such as at least 30, 40,50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 750,1000 or more contiguous nucleotides from the reference nucleotidesequence, up to the full length of the reference nucleotide sequence.The oligonucleotides of the invention are thus of sufficient length tobe useful as sequencing primers, PCR primers, hybridization probes orantisense reagents, and can also encode polypeptides having 2,5-DKGpermease activity, or immunogenic peptides therefrom. Those skilled inthe art can determine the appropriate length and sequence of anoligonucleotide of the invention for a particular application.

[0082] For certain applications, such as for detecting 2,5-DKGexpression in a cell or library, it will be desirable to use isolatedoligonucleotide molecules of the invention that specifically hybridizeto a nucleic acid molecule encoding a 2,5-DKG permease. The term“specifically hybridize” refers to the ability of a nucleic acidmolecule to hybridize, under stringent hybridization conditions asdescribed below, to a nucleic acid molecule that encodes a 2,5-DKGpermease, without hybridizing to a substantial extent under the sameconditions with nucleic acid molecules that do not encode 2,5-DKGpermeases, such as unrelated molecules that fortuitously contain shortregions of identity with a permease sequence. Thus, a nucleic acidmolecule that “specifically hybridizes” is of a sufficient length andcontains sufficient distinguishing sequence from a 2,5-DKG permease tobe characteristic of the 2,5-DKG permease. Such a molecule willgenerally hybridize, under stringent conditions, as a single band on aNorthern blot or Southern blot prepared from mRNA of a single species.

[0083] As used herein, the term “stringent conditions” refers toconditions equivalent to hybridization of a filter-bound nucleic acidmolecule to a nucleic acid in a solution containing 50% formamide, 5×Denhart's solution, 5× SSPE, 0.2% SDS at 42° C., followed by washing thefilter in 0.1× SSPE, and 0.1% SDS at 65° C. twice for 30 minutes.Equivalent conditions to the stringent conditions set forth above arewell known in the art, and are described, for example in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1992).

[0084] Nucleotide sequences that are characteristic of each of SEQ IDNOS: 1, 3, 5, 7 or 9, or which are common to two, three or more of SEQID NOS: 1, 3, 5, 7, 9 or 11 can readily be determined by aligning thesequences using a CLUSTAL V alignment program. Oligonucleotidescontaining regions which are common to two or more different 2,5-DKGpermease nucleic acid molecules can advantageously be used as PCRprimers or hybridization probes to isolate or detect nucleic acidmolecules encoding 2,5-DKG permeases from other species.

[0085] The oligonucleotides of the invention can, but need not, encodepolypeptides having 2,5-DKG activity. Thus, the inventionoligonucleotides can contain sequences from the 5′ or 3′ untranslatedregion, or both, of the nucleotide sequences designated SEQ ID NOS: 1,3, 5, 7, 9 or 11, or contain coding sequences, or both. As describedabove with respect to the term “nucleic acid molecule,” the inventionoligonucleotides can be derived from either the sense or antisensestrand of the recited SEQ ID NO.

[0086] The oligonucleotides of the invention can also advantageously beused to direct the incorporation of amino acid additions, deletions orsubstitutions into a recombinant 2,5-DKG permease. In such applications,it will be understood that the invention oligonucleotides can containnucleotide modifications with respect to SEQ ID NOS: 1, 3, 5, 7, 9 or 11such that the oligonucleotides encode the desired amino acidmodifications to SEQ ID NOS: 2, 4, 6, 8, 10 or 12, so long as theycontain at least 17 contiguous residues from the reference sequence.

[0087] Exemplary oligonucleotides of the invention are oligonucleotidesthat contain a sequence selected from nucleotides 1-20, 1-100, 101-120,101-200, 201-220, 201-300, 301-320, 301-400, 401-420, 401-500, 501-520,501-600, 601-620, 601-700, 701-720, 701-800, 801-820, 801-900, 901-920,901-1000, 1001-1020, 1001-1100, 1101-1120, 1100-1200, 1201-1220,1201-1300, 1301-1320, 1301-1400, 1401-1420 or 1401-1500 of any of SEQ IDNOS: 1, 3, 5, 7, 9 or 11.

[0088] The invention further provides a kit containing a pair of 2,5-DKGpermease oligonucleotides packaged together, either in a singlecontainer or separate containers. The pair of oligonucleotides arepreferably suitable for use in PCR applications for detecting oramplifying a nucleic acid molecule encoding a 2,5-DKG permease. The kitcan further contain written instructions for use of the primer pair inPCR applications, or solutions and buffers suitable for suchapplications.

[0089] The invention further provides isolated oligonucleotides thatcontain a nucleotide sequence encoding a peptide having at least 10contiguous amino acids of an amino acid selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10 or 12. Such oligonucleotidescan encode at least 10, 12, 15, 20, 25 or more contiguous amino acids ofSEQ ID NOS: 2, 4, 6, 8, 10 or 12, such as at least 30, 40, 50, 75, 100,200, 300, 400 or more contiguous amino acids from the referencesequence. The encoded peptides can be expressed from sucholigonucleotides, by routine methods, and used to produce, purify orcharacterize 2,5-DKG antibodies, as will be discussed further below. Thepeptides encoded by such oligonucleotides can, but need not,additionally have 2,5-DKG permease enzymatic activity.

[0090] In one embodiment, the isolated oligonucleotide encodes an aminoacid sequence selected from amino acids 1-10, 1-50, 51-60, 51-100,101-110, 101-150, 151-160, 151-200, 201-210, 201-250, 251-260, 251-300,301-310, 301-350, 351-361, 351-400, 401-410, 401-439 of any of SEQ IDNOS: 2, 4, 6, 8, 10 or 12.

[0091] Isolated nucleic acid molecules which encode polypeptides having2,5-DKG permease activity, as well as the isolated oligonucleotidesdescribed above, will be subsequently referred as “2,5-DKG permeasenucleic acid molecules.”

[0092] The isolated 2,5-DKG permease nucleic acid molecules of theinvention can be prepared by methods known in the art. The method chosenwill depend on factors such as the type and size of nucleic acidmolecule one intends to isolate; whether or not it encodes abiologically active polypeptide (e.g. a polypeptide having permeaseactivity or immunogenicity); and the source of the nucleic acidmolecule. Those skilled in the art can isolate or prepare 2,5-DKGpermease nucleic acid molecules as genomic DNA or desired fragmentstherefrom; as full-length cDNA or desired fragments therefrom; or asfull-length mRNA or desired fragments therefrom, from any microorganismof interest.

[0093] An exemplary method of preparing a 2,5-DKG permease nucleic acidmolecule is by isolating a recombinant construct which encodes andexpresses a polypeptide having 2,5-DKG permease activity. As describedin the Example, one useful method is to provide a metabolic selectionsystem where bacterial cell growth is made dependent on expression of a2,5-DKG permease, introducing expressible DNA, such as a cDNA or genomiclibrary, into the assay cells, selecting surviving cells under theselective conditions, and isolating the introduced DNA. Alternatively, ascreening method can be designed, such that a cell will exhibit adetectable signal only when expressing a functional 2,5-DKG permease. Anexemplary detectable signal is intracellular incorporation of adetectable label present on 2,5-DKG. Additionally screening andselection strategies suitable for identifying nucleic acid moleculesencoding metabolic enzymes are described, for example, in PCTpublication WO 00/22170 and U.S. Pat. Nos. 5,958,672 and 5,783,431.

[0094] A further method for producing an isolated 2,5-DKG permeasenucleic acid molecule involves amplification of the nucleic acidmolecule using 2,5-DKG permease-specific primers and the polymerasechain reaction (PCR). Using PCR, a 2,5-DKG permease nucleic acidmolecule having any desired boundaries can be amplified exponentiallystarting from as little as a single gene or mRNA copy, from any cellhaving a 2,5-DKG permease gene.

[0095] Given the high degree of identity among the six disclosed 2,5-DKGpermeases, those skilled in the art can design suitable primers forisolating additional 2,5-DKG permease nucleic acid molecules. Suchprimers are preferably degenerate oligonucleotides that encode, or arecomplementary to, short consensus amino acid sequences present in two ormore of the 2,5-DKG permeases disclosed herein, such as oligonucleotidesthat encode 10 or more contiguous amino acids present in at least two ofSEQ ID NOS: 2, 6 and 10, or oligonucleotides that encode 10 or morecontiguous amino acids present in at least two of SEQ ID NOS: 4, 8 and12. Such sequences can be determined from an alignment of amino acidsequences shown in FIG. 1. Exemplary amino acid sequences present in atleast two of SEQ ID NOS: 2, 6 and 10 are amino acids 19-31, 115-124,146-156, and 339-348 of SEQ ID NO: 2. Exemplary amino acid sequencespresent in at least two of SEQ ID NOS: 4, 8 and 12 are amino acids55-64, 60-69, 252-261, and 370-379 of SEQ ID NO: 8.

[0096] Methods are well known in the art to determine or modify PCRreaction conditions when using degenerate primers to isolate a desirednucleic acid molecule. The amplified product can subsequently besequenced, used as a hybridization probe, or used for 5′ or 3′ RACE toisolate flanking sequences, following procedures well known in the artand described, for example, in Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York (2000).

[0097] Given the high degree of sequence identity and structuralrelatedness among the six disclosed 2,5-DKG permeases, homologs from anyother species can readily be identified by either hybridization orantibody screening. For example, an isolated 2,5-DKG permease nucleicacid molecule can be identified by screening a library, such as agenomic library, cDNA library or expression library, with a detectablenucleic acid molecule or antibody. Such libraries are commerciallyavailable from a variety of microorganisms, or can be produced from anyavailable microorganism or environmental sample of interest usingmethods described, for example, in PCT publication WO 00/22170. Thelibrary clones identified as containing 2,5-DKG permease nucleic acidmolecules can be isolated, subcloned and sequenced by routine methods.

[0098] Furthermore, 2,5-DKG permease nucleic acid molecules can beproduced by direct synthetic methods. For example, a single strandednucleic acid molecule can be chemically synthesized in one piece, or inseveral pieces, by automated synthesis methods known in the art. Thecomplementary strand can likewise be synthesized in one or more pieces,and a double-stranded molecule made by annealing the complementarystrands. Direct synthesis is particularly advantageous for producingrelatively short molecules, such as oligonucleotide probes and primers,and also for producing nucleic acid molecules containing modifiednucleotides or linkages.

[0099] The invention also provides an isolated polypeptide which has2,5-DKG permease activity. Such isolated polypeptides, when expressed intheir normal configuration at the cell membrane, are useful inapplications in which enhanced uptake of 2,5-DKG is desirable, such asin bioproduction of 2-KLG. The isolated polypeptides of the inventioncan also be added to a culture medium, preferably in a membrane vesicle,to compete with membrane-bound permeases for 2,5-DKG, and thus to stop2,5-DKG uptake. Thus, isolated polypeptides having 2,5-DKG permeaseactivity can be used to regulate production of 2,5-DKG metabolites.

[0100] In one embodiment, an isolated polypeptide of the invention isnot encoded by a nucleotide sequence completely contained within thenucleotide sequence designated SEQ ID NO: 19 of WO 00/22170, which isthe K. oxytoca yia operon. In another embodiment, an isolatedpolypeptide of the invention is not completely contained within theamino acid sequence herein designated SEQ ID NO: 12.

[0101] An “isolated” polypeptide of the invention is altered by the handof man from how it is found in its natural environment. For example, anisolated 2,5-DKG permease can be a molecule that is recombinantlyexpressed, such that it is present at a higher level in its native host,or is present in a different host. Alternatively, an “isolated” 2,5-DKGpermease of the invention can be a substantially purified molecule.Substantially purified 2,5-DKG permeases can be prepared by methodsknown in the art. Specifically with respect to a polypeptide encodingthe yiaX2 polypeptide designated SEQ ID NO: 12, the term “isolated” isintended to mean that polypeptide is not present in association with thepolypeptides expressed by other genes in the K. oxytoca yia operon, suchas the genes designated lyxK and orf1, described in WO 00/22170.

[0102] In one embodiment, an isolated polypeptide having 2,5-DKGpermease activity contains an amino acid sequence having at least 40%identity to an amino acid sequence selected from the group consisting ofSEQ ID NOS: 2, 4, 6, 8, 10 or 12. Preferably, the encoded polypeptidewill have at least 45% identity to any of the recited SEQ ID NOS, suchas at least 50%, 60%, 70%, 80% identity, including at least 90%, 95%,98%, 99% or greater identity.

[0103] In another embodiment, the isolated polypeptide having 2,5-DKGpermease activity contains at least 10 contiguous amino acids of any ofSEQ ID NOS: 2, 4, 6, 8, 10 or 12. Exemplary invention polypeptidescontain an amino acid sequence of amino acids 1-10, 1-50, 51-60, 51-100,101-110, 101-150, 151-160, 151-200, 201-210, 201-250, 251-260, 251-300,301-310, 301-350, 351-361, 351-400, 401-410, 401-439 of any of SEQ IDNOS: 2, 4, 6, 8, 10 or 12.

[0104] Also provided is an isolated immunogenic peptide having an aminoacid sequence derived from a 2,5-DKG permease. Such isolated immunogenicpeptides are useful, for example, in preparing and purifying 2,5-DKGantibodies. The term “immunogenic,” as used herein, refers to a peptidethat either is capable of inducing 2,5-DKG permease-specific antibodies,or capable of competing with 2,5-DKG permease-specific antibodies forbinding to a 2,5-DKG permease. Peptides that are likely to beimmunogenic can be predicted using methods and algorithms known in theart and described, for example, by Irnaten et al., Protein Eng.11:949-955 (1998), and Savoie et al., Pac. Symp. Biocomput. 1999:182-189(1999). The immunogenicity of the peptides of the invention can beconfirmed by methods known in the art, such as by delayed-typehypersensitivity response assays in an animal sensitized to a 2,5-DKGpermease, or by direct or competitive ELISA assays.

[0105] An isolated immunogenic peptide of the invention can contain atleast 10 contiguous amino acids of a polypeptide selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10 or 12, such as amino acids1-10, 1-50, 51-60, 51-100, 101-110, 101-150, 151-160, 151-200, 201-210,201-250, 251-260, 251-300, 301-310, 301-350, 351-361, 351-400, 401-410,401-439 of any of SEQ ID NOS: 2, 4, 6, 8, 10 or 12. Such a peptide canhave at least 12, 15, 20, 25 or more contiguous amino acids of thereference sequence, including at least 30, 40, 50, 75, 100, 200, 300,400 or more contiguous amino acids from the reference sequence, up tothe full-length sequence.

[0106] For the production of antibodies that recognize 2,5-DKG permeasesin their native configuration, such peptides will preferably contain atleast part of an extracellular or intracellular domain from thepermease. An extracellular or intracellular domain is generallycharacterized by containing at least one polar or positively ornegatively charged residue, whereas a transmembrane domain is generallycharacterized as an uninterrupted stretch of about 20 contiguoushydrophobic residues. Commercially available computer topology programscan be used to determine whether a peptide is likely to correspond to anextracellular or intracellular domain or to a transmembrane region.Immunogenic peptides of the invention derived from a transmembraneregion are useful to raise antibodies for use in applications such asimmunoblotting, where the 2,5-DKG polypeptide need not be in its nativeconfiguration to be recognized.

[0107] The structural and functional characteristics and applications of2,5-DKG permease polypeptides of the invention have been described abovewith respect to the encoding nucleic acid molecules, and are equallyapplicable in reference to the isolated polypeptides of the invention.Isolated polypeptides having 2,5-DKG permease activity, as well as theisolated immunogenic peptides of the invention, will subsequently bereferred to as “2,5-DKG permeases.”

[0108] Methods for recombinantly producing 2,5-DKG permeases have beendescribed above with respect to nucleic acid molecules, vectors andcells of the invention. 2,5-DKG permeases can alternatively be preparedby biochemical procedures, by isolating membranes from bacteria thatnaturally express, or recombinantly express, 2,5-DKG permeases. Themembranes can be further fractionated by size or affinitychromatography, electrophoresis, or immunoaffinity procedures, toachieve the desired degree of purity. Purification can be monitored by avariety of procedures, such as by immunoreactivity with 2,5-DKG permeaseantibodies, or by a functional assay.

[0109] Immunogenic peptides can be produced from purified or partiallypurified 2,5-DKG permease polypeptides, for example, by enzymatic orchemical cleavage of the full-length polypeptide. Methods for enzymaticand chemical cleavage and for purification of the resultant peptidefragments are well known in the art (see, for example, Deutscher,Methods in Enzymology, Vol. 182, “Guide to Protein Purification,” SanDiego: Academic Press, Inc. (1990)).

[0110] Alternatively, 2,5-DKG permeases can be produced by chemicalsynthesis. If desired, such as to optimize their functional activity,stability or bioavailability, such chemically synthesized molecules caninclude D-stereoisomers, non-naturally occurring amino acids, and aminoacid analogs and mimetics. Sawyer, Peptide Based Drug Design, ACS,Washington (1995) and Gross and Meienhofer, The Peptides: Analysis,Synthesis. Biology, Academic Press, Inc., New York (1983). For certainapplications, such as for detecting the polypeptide, it can also beuseful to incorporate one or more detectably labeled amino acids into achemically synthesized permease, such as radiolabeled or fluorescentlylabeled amino acids.

[0111] An isolated 2,5-DKG permease of the invention can further beconjugated to carrier molecules, such as keyhole lympet hemocyanin,which can enhance recognition by the immune system of the isolated2,5-DKG permease for production of antibodies. For certain applications,such as to increase the stability or bioactivity of the molecule, or tofacilitate its identification, the 2,5-DKG permease can be chemically orenzymatically derivatized, such as by acylation, phosphorylation orglycosylation.

[0112] The invention also provides an antibody specific for apolypeptide having 2,5-DKG permease activity, such as an antibodyspecific for a polypeptide having the amino acid sequence of any of SEQID NOS: 2, 4, 6, 8, 10 or 12. Also provided is an antibody specific foran isolated peptide that contains at least 10 contiguous amino acids ofany of SEQ ID NOS: 2, 4, 6, 8, 10 or 12, wherein the peptide isimmunogenic. The antibodies of the invention can be used, for example,to detect or isolate 2,5-DKG permeases from expression libraries orcells.

[0113] The term “antibody,” as used herein, is intended to includemolecules having specific binding activity for a 2,5-DKG permease of atleast about 1×10⁵ M⁻¹, preferably at least 1×10⁷ M⁻¹, more preferably atleast 1×10⁹ M⁻¹. The term “antibody” includes both polyclonal andmonoclonal antibodies, as well as antigen binding fragments of suchantibodies (e.g. Fab, F(ab′)₂, Fd and Fv fragments and the like). Inaddition, the term “antibody” is intended to encompass non-naturallyoccurring antibodies, including, for example, single chain antibodies,chimeric antibodies, bifunctional antibodies, CDR-grafted antibodies andhumanized antibodies, as well as antigen-binding fragments thereof.

[0114] Methods of preparing and isolating antibodies, includingpolyclonal and monoclonal antibodies, using peptide and polypeptideimmunogens, are well known to those skilled in the art and aredescribed, for example, in Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press (1988). Non-naturallyoccurring antibodies can be constructed using solid phase peptidesynthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains. Such methods are described, forexample, in Huse et al. Science 246:1275-1281 (1989); Winter and Harris,Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546(1989); Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); and Borrabeck, Antibody Engineerinq, 2d ed. (OxfordUniversity Press 1995).

[0115] The following example is intended to illustrate but not limit thepresent invention.

EXAMPLE

[0116] This example shows the isolation and characterization of nucleicacid molecules encoding six novel polypeptides having 2,5-DKG permeaseactivity.

[0117] Identification of yiaX2 as a 2,5-DKG Permease

[0118] WO 002170 describes the identification and sequencing of anoperon from Klebsiella oxytoca, designated the yia operon, whichcontains eight putative open reading frames. Because disruption of thisoperon abolished the ability of K. oxytoca to utilize ascorbic acid asthe sole carbon source, the yia operon was predicted to be involved inthe catabolism of ascorbic acid. The functions of the polypeptidesencoded by the individual open reading frames in the yia operon were notdescribed in WO 002170.

[0119] It was determined that K. oxytoca was able to grow on 2,5-DKG asa sole carbon source and, therefore, it was concluded that K. oxytocaexpressed a 2,5-DKG permease. It was predicted that such a permeasewould share structural properties with known bacterial transporterproteins, such as multiple transmembrane segments. One of theuncharacterized open reading frames in the yia operon, designated yiaX2,encoded a transmembrane polypeptide with about 33% identity to a knowntartrate transporter, and was thus considered a candidate 2,5-DKGpermease.

[0120] In order to determine whether yiaX2 encoded a 2,5-DKG permease,this gene was deleted from the chromosome of the K. oxytoca straindesignated M5a1. M5a1 has also been described in the literature as K.pneumonia (see, for example, Streicher et al., Proc. Natl. Acad. Sci.68:1174-1177 (1971)). The yiaX2 deletion mutant was constructed byjoining sequences immediately upstream and downstream of the yiaX2 genein a three-way ligation with the pMAK705 integration vector (describedin Hamilton et al., J. Bacteriol. 171:4617-4622 (1989)). A fragment ofabout 1 kb in the orf1 gene was amplified using oligonucleotides5′-ACCCAAGCTTCACCAAAAGAGTGAAGAGGAAG-3′ (SEQ ID NO: 17) and5′-CGTATCTAGAAAAATATTCTGGTGATGAAGGTGA-3 (SEQ ID NO: 18), and digestedwith HindIII and XbaI. A fragment of a similar size in the lyxK gene wasamplified with oligonucleotides 5′-AGACTCTAGATCCACATAAACGCACTGCGTAAAC-3′(SEQ ID NO: 19) and 5′-GAGGGGATCCTGGCTTCGTGAACGATATACTGG-3′ (SEQ ID NO:20), and digested with XbaI and BamHI. The two resulting fragments wereligated together between the HindIII and BamHI sites of the vectorpMAK705. The resulting plasmid was transformed into K. oxytoca strainM5a1, and candidates in which the deletion construct had integrated bydouble crossover were obtained as described in Hamilton et al., supra(1989). The designation of the resulting K. oxytoca ΔyiaX2 strain isMGK002. The yiaX2-deficient phenotype was verified by by PCR analysis.

[0121] As described below, the K. oxytoca ΔyiaX2 [tkr idnO] strain wasdetermined to grow very inefficiently on 2,5-DKG as the sole carbonsource, and not to grow on 2-KLG. Confirmation that yiaX2 encoded a 30polypeptide having 2,5-DKG and 2-KLG permease activities was obtained bydetermining that adding back the gene restored the ability of the K.oxytoca ΔyiaX2 [tkr idnO] to grow well on either 2,5-DKG or 2-KLG (seebelow).

[0122] Construction of K. oxytoca ΔviaX2 [tkr idnO]

[0123] In order to identify additional 2,5-DKG permeases, and preferablypermeases selective for 2,5-DKG, a metabolic selection strategy wasutilized. As described in WO 00/22170, metabolic selection isadvantageous in allowing rapid identification of functional genes fromuncharacterized and even unculturable microorganisms, without any priorsequence information.

[0124] A tester strain for the metabolic selection of nucleic acidmolecules encoding 2,5-DKG permeases was prepared by engineering K.oxytoca ΔyiaX2 to express enzymes involved in the catabolism of 2,5-DKGto gluconic acid, which can be converted to carbon and energy. Enzymescapable of catabolizing 2,5-DKG to gluconic acid are encoded by the tkrand idnO genes of the tkr idnD idnO operon designated SEQ ID NO: 13.

[0125] The tkr idnD idnO operon (SEQ ID NO: 13) was subcloned into thehigh copy number vector pUC19 and the resulting clone, designated pDF33,was transformed into K. oxytoca ΔyiaX2. The resulting tester strain(designated MGK002 [pDF33] or K. oxytoca ΔyiaX2 [tkr idnO]) thusexpresses all polypeptides required for the utilization of 2,5-DKG as asole carbon source, but is deficient in 2,5-DKG permease activity totransport extracellular 2,5-DKG into the cell. Therefore, a nucleic acidmolecule that encodes a 2,5-DKG permease, upon expression in the testerstrain, should confer the ability of the tester strain to grow on2,5-DKG. The metabolic selection strategy is shown schematically in FIG.3.

[0126] To validate the proposed metabolic selection strategy, as apositive control the yiaX2 gene was reintroduced into the tester strainto confirm that it conferred the ability to grow on 2,5-DKG and 2-KLG.The yiaX2 open reading frame (nucleotides 3777 to 5278 of SEQ ID NO: 19of WO 00/22170) was PCR-amplified using olignucleotides5′-AATAGGATCCTTCATCACCAGAATATTTTTA-3′ (SEQ ID NO: 21) and5′-CATAGGTACCGGCTTTCAGATAGGTGCC-3′ (SEQ ID NO: 22) digested with BamH1and Kpn1 and ligated into pCL1920 (Lerner et al., Nucl. Acids. Res.18:4631 (1990); and see description below) previously digested with thesame restriction enzymes. K. oxytoca ΔyiaX2 [tkr idnO], transformed withthe resulting construct, was able to grow overnight at 30° C. on M9minimal agar medium supplemented with either 2-KLG or 2,5-DKG (0.25%)and 0.1 mM IPTG. Therefore, K. oxytoca ΔyiaX2 [tkr idnO] was confirmedto be an appropriate tester strain to identify additional novel 2,5-DKGpermeases, and to determine their selectivity.

[0127] Construction of Bacterial Genomic Libraries

[0128] The cloning vector used for constructing the above positivecontrol and for preparing bacterial genomic libraries is plasmid pCL1920(Lerner et al., supra, 1990), a low-copy number expression vector whichcarries a spectinomycin/streptomycin resistance determinant. Expressionis driven by the lacPO promoter/operator region which is repressed bythe lacI^(q) gene product when provided by the host, and induced in thepresence of 0.01 to 1 mM IPTG.

[0129] Genomic DNA from the following species and isolates was preparedaccording to the method outlined below: Pantoea citrea (ATCC 39140),Klebsiella oxytoca MGK002 (ΔyiaX2), Pseudomonas aeruginosa, and amixture of 25 environmental isolates, obtained from 18 different soiland water samples, and able to grow on 2,5-DKG as the sole carbonsource. Klebsiella oxytoca MGK002 (ΔyiaX2) was among the bacteria chosenbecause there was a slight amount of background growth observable in thetester strain on 2,5-DKG as the sole carbon source, and some 2,5-DKGpermease activity in an uptake assay. However, the tester strain did notgrow on 2-KLG, and exhibited no detectable 2-KLG uptake, suggesting thepresence of a second 2,5-DKG permease with selectivity for 2,5-DKG in K.oxytoca.

[0130] Five milliliters of an overnight culture in LB (30° C.) werecentrifuged for 5 min at 6,000 rpm. Pellets were washed with 1.5 ml Tris10 mM, EDTA 1 mM pH 8.0 (TE), centrifuged again and resuspended in 0.4ml TE. Lysozyme (5 mg/ml) and RNase (100 pg/ml) were added and cellswere incubated for 10 min at 37° C. Sodium dodecylsulfate (SDS) wasadded to a final concentration of 1% and the tubes were gently shakenuntil lysis was complete. One hundred microliters of a 5N NaCl0₄ stocksolution were added to the lysate. The mixture was extracted once withone volume of phenol:chloroform (1:1) and once with one volume ofchloroform. Chromosomal DNA was precipitated by adding 2 ml of cold(−20° C.) ethanol and gently coiling the precipitate around a curvedPasteur pipette. DNA was dried for 30 min at room temperature andresuspended in 50 to 100 μl of Tris 10 mM, EDTA 1 mM, NaCl 50 mM pH 8.0to obtain a DNA concentration of 0.5 to 1 μg/μl. Genomic DNApreparations from each environmental isolate were mixed in equal ratiosto prepare a single mixed library.

[0131] For each preparation, an aliquot of 10-15 μl of genomic DNA wassubjected to Sau3A controlled digestion in order to obtain fragmentsranging between 3 to 20 kb in size. Half that amount was ligated withthe low-copy number expression vector pCL1920, which had previously beendigested with BamHI and dephosphorylated. The resulting genomiclibraries were transformed into E. coli DH10B electrocompetent cells(GIBCO-BRL) and briefly amplified overnight at 30° C. on LB-agarsupplemented with 100 μg/ml spectinomycin. For each library, 30,000 to120,000 clones were plated out and plasmid DNA was bulk-extracted usingstandard procedures. Insert size was randomly checked and the amplifiedlibraries were stored in the form of plasmid DNA at −20° C. for furtheruse in the tester strain.

[0132] Selection, Identification and Sequencing of Permease Genes

[0133] An aliquot of each genomic library was introduced byelectroporation into the K. oxytoca ΔyiaX2 [tkr idnO] (MGK002[pDF33])strain. The amount of DNA used in the transformation was adjusted inorder to plate out 5×10⁵ to 1×10⁶ clones per library on the selectivemedium. Each selection round was plated on LB-agar containing 100 μg/mlspectinomycin, then replica-plated onto M9-agar plates containing 2.5%2,5-DKG and 0.1 mM IPTG and adjusted to pH 4.5. The clones that grew on2,5-DKG were transferred into K. oxytoca ΔyiaX2 (MGK002) devoid ofplasmid pDF33, to verify that the tkr idnDO pathway was indeed requiredfor growth of those clones on 2,5-DKG. A brief genetic characterizationwas performed to eliminate identical clones. Following preliminary2,5-DKG/ 2-KLG uptake assays, 5 clones were retained for furtheranalysis: 2 originated from the Pantoea citrea library, 1 from K.oxytoca and 2 from the mixed environmental library.

[0134] In all cases, DNA sequencing of the vector inserts revealed thepresence of a nucleotide sequence (SEQ ID NOS: 1, 3, 5, 7 and 9)encoding a polypeptide (SEQ ID NOS: 2, 4, 6, 8 and 10) displayinghomology with published transporters and with yiaX2 (SEQ ID NO: 11, andits encoded polypeptide SEQ ID NO: 12). Also present on these insertswere other orfs, and in most cases an endogenous promoter.

[0135] The insert containing both of the prmA and prmB orfs (SEQ ID NOS:7 and 9) was about 9 kb, and also contained an orf homologous tobacterial idnO, two orfs encoding transcriptional repressors, an orf ofunknown function, and 3 orfs encoding homologs of E. coli polypeptidesinvolved in nitrate utilization.

[0136] The insert containing the PE1 orf (SEQ ID NO: 1) was about 3 kb,and also contained a putative dehydro-deoxygluconokinase gene closelyrelated to the B. subtilis kdgK gene and a homolog of the E. coli ydcGgene.

[0137] The insert containing the PE6 orf (SEQ ID NO: 3) was about 6.7kb. The genomic environment of PE6 appeared similar to the yia operon ofE. coli and K. oxytoca, as SEQ ID NO: 4 was preceded by a yiaL homologand a yiaK homolog was also present on the insert.

[0138] The insert containing the PK1 orf (SEQ ID NO: 5) was about 5.5kb. In contrast to the other inserts, this insert did not appear tocontain an endogenous promoter, indicating that the PK1 orf wasapparently transcribed from the vector's promoter. The PK1 orf wasdirectly followed by a tkr homolog.

[0139] Nucleic acid molecules encoding each 2,5-DKG permease werereintroduced into K. oxytoca ΔyiaX2 [tkr idnO] and the resulting strainsassayed for growth on 2,5-DKG and 2-KLG, and also assayed for uptake ofradiolabeled 2,5-DKG and 2-KLG.

[0140] The uptake assays were performed by mixing radioactive 2-KLG or2,5-DKG with IPTG-induced cells, removing aliquots at regular intervals,and measuring both the decrease in radioactivity in the supernatant andthe appearance of radioactivity in the cells over time. The results ofthe growth assays and 2,5-DKG uptake assay are shown in Table 1, below.TABLE 1 Recombinantly 2,5-DKG expressed Cell Growth Cell Growth Uptake2,5 DKG Permease on 2,5-DKG on 2-KLG (g/l/h) YiaX2 + ++ 3.7 PE1 ++ ++4.2 PE6 ++ ++ 5.0 prmA ++ ++ 5.5 prmB +/− ND 0.9 prmA and prmB ++ ND 9.9PK1 ++ — 4.2 Control bkgd — 1.0 (K. oxytoca ΔyiaX2/ tkr/idn0)

[0141] Nucleic acid molecules encoding the different permeases were alsosubcloned into a variety of vectors, including the high copy numbervector pSE380 (which contains a tac promoter), the medium copy numbervector pACYC184 (which is promoterless), or the low copy number vectorpCL1920, and introduced into a Pantoea strain suitable for bioproductionof 2-KLG from glucose (see U.S. Pat. No. 5,032,514). The resultingstrains were assessed under biofermentation conditions to determinewhich combinations of nucleic acid molecules, promoters and vectors areoptimal for enhancing 2-KLG production.

[0142] All journal article, reference and patent citations providedabove, in parentheses or otherwise, whether previously stated or not,are incorporated herein by reference in their entirety.

[0143] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1 22 1 1500 DNA Unknown environmental source 1 ggcgaatagc ccggccggcgtcataataac ggccttctct gtaccctaca tacggcggcg 60 gcgtcatgaa cctcaactttagtaggcaag cct atg aac agc tct acc aat gca 114 Met Asn Ser Ser Thr AsnAla 1 5 acg aaa cgc tgg tgg tac atc atg cct atc gtg ttt atc acg tat agc162 Thr Lys Arg Trp Trp Tyr Ile Met Pro Ile Val Phe Ile Thr Tyr Ser 1015 20 ctg gcg tat ctc gac cgc gca aac ttc agc ttt gct tcg gca gcg ggc210 Leu Ala Tyr Leu Asp Arg Ala Asn Phe Ser Phe Ala Ser Ala Ala Gly 2530 35 att acg gaa gat tta ggc att acc aaa ggc atc tcg tcg ctt ctt ggc258 Ile Thr Glu Asp Leu Gly Ile Thr Lys Gly Ile Ser Ser Leu Leu Gly 4045 50 55 gca ctt ttc ttc ctc ggc tat ttc ttc ttc cag atc ccg ggg gcg att306 Ala Leu Phe Phe Leu Gly Tyr Phe Phe Phe Gln Ile Pro Gly Ala Ile 6065 70 tac gcg gaa cgc cgt agc gta cgg aag ctg att ttc atc tgt ctg atc354 Tyr Ala Glu Arg Arg Ser Val Arg Lys Leu Ile Phe Ile Cys Leu Ile 7580 85 ctg tgg ggc gcc tgc gcc tcg ctt gac cgg gat ggt gca caa tat tcc402 Leu Trp Gly Ala Cys Ala Ser Leu Asp Arg Asp Gly Ala Gln Tyr Ser 9095 100 agc gct ggc tgg cga tcc gtt tta ttc tcg gct gtc gtg gaa gcg gcg450 Ser Ala Gly Trp Arg Ser Val Leu Phe Ser Ala Val Val Glu Ala Ala 105110 115 gtc atg ccg gcg atg ctg att tac atc agt aac tgg ttt acc aaa tca498 Val Met Pro Ala Met Leu Ile Tyr Ile Ser Asn Trp Phe Thr Lys Ser 120125 130 135 gaa cgt tca cgc gcc aac acc ttc tta atc ctc ggc aac ccg gtcacg 546 Glu Arg Ser Arg Ala Asn Thr Phe Leu Ile Leu Gly Asn Pro Val Thr140 145 150 gta ctg tgg atg tcg gtg gtc tcc ggc tac ctg att cag tcc ttcggc 594 Val Leu Trp Met Ser Val Val Ser Gly Tyr Leu Ile Gln Ser Phe Gly155 160 165 tgg cgt gaa atg ttt att att gaa ggc gtt ccg gcc gtc ctc tgggcc 642 Trp Arg Glu Met Phe Ile Ile Glu Gly Val Pro Ala Val Leu Trp Ala170 175 180 ttc tgc tgg tgg gtg ctg gtc aaa gtt aaa ccg tcg cag gtg aactgg 690 Phe Cys Trp Trp Val Leu Val Lys Val Lys Pro Ser Gln Val Asn Trp185 190 195 ttg tcg gaa aac gag aaa gcc gcg ctg cag gcg cag ctg gag agcgag 738 Leu Ser Glu Asn Glu Lys Ala Ala Leu Gln Ala Gln Leu Glu Ser Glu200 205 210 215 cag cag ggt att aaa gcc gtg cgt aac tac ggc gaa gcc ttccgc tca 786 Gln Gln Gly Ile Lys Ala Val Arg Asn Tyr Gly Glu Ala Phe ArgSer 220 225 230 cgc aac gtc att cta ctg tgc atg cag tat ttt gcc tgg agtatc ggc 834 Arg Asn Val Ile Leu Leu Cys Met Gln Tyr Phe Ala Trp Ser IleGly 235 240 245 gtg tac ggt ttt gtg ctg tgg ttg ccg tca att att cgc agcggc ggc 882 Val Tyr Gly Phe Val Leu Trp Leu Pro Ser Ile Ile Arg Ser GlyGly 250 255 260 gtc aat atg ggg atg gtg gaa gtc ggc tgg ctc tct tcg gtgcct tat 930 Val Asn Met Gly Met Val Glu Val Gly Trp Leu Ser Ser Val ProTyr 265 270 275 ctg gcc gcg act att gcg atg atc gtc gtc tcc tgg gct tccgat aaa 978 Leu Ala Ala Thr Ile Ala Met Ile Val Val Ser Trp Ala Ser AspLys 280 285 290 295 atg cag aac cgt aaa ctg ttc gtc tgg ccg ctg ctg ctgatt ggc gga 1026 Met Gln Asn Arg Lys Leu Phe Val Trp Pro Leu Leu Leu IleGly Gly 300 305 310 ctg gct ttt att ggc tca tgg gcc gtc ggc gct aac catttc tgg gcc 1074 Leu Ala Phe Ile Gly Ser Trp Ala Val Gly Ala Asn His PheTrp Ala 315 320 325 tct tat acc ctg ctg gtg att gcc aat gcg gca atg tacgcc cct tac 1122 Ser Tyr Thr Leu Leu Val Ile Ala Asn Ala Ala Met Tyr AlaPro Tyr 330 335 340 ggt ccg ttt ttc gcc atc att ccg gaa atg ctg ccg cgtaac gtc gcc 1170 Gly Pro Phe Phe Ala Ile Ile Pro Glu Met Leu Pro Arg AsnVal Ala 345 350 355 ggt ggc gca atg gcg ctc atc aac agc atg ggg gcc ttaggt tca ttc 1218 Gly Gly Ala Met Ala Leu Ile Asn Ser Met Gly Ala Leu GlySer Phe 360 365 370 375 ttt ggt tcg tgg ttc gtg ggc tac ctg aac ggc accacc ggc agt cca 1266 Phe Gly Ser Trp Phe Val Gly Tyr Leu Asn Gly Thr ThrGly Ser Pro 380 385 390 tca gcc tca tac att ttc atg gga gtg gcg ctt ttcgcc tcg gta tgg 1314 Ser Ala Ser Tyr Ile Phe Met Gly Val Ala Leu Phe AlaSer Val Trp 395 400 405 ctt act tta att gtt aag cct gct aac aat caa aagctc ccc atc ggc 1362 Leu Thr Leu Ile Val Lys Pro Ala Asn Asn Gln Lys LeuPro Ile Gly 410 415 420 gct cgt cac gcc tgacctttac tacttacgga gatcacgccttgggtacgtt 1414 Ala Arg His Ala 425 gcaggacaaa ccgataggca ccgcaaaggctggggccatc gagcagcgcg taaacagtca 1474 gctggttgct gtcgctgtgc ggcgtc 15002 427 PRT Unknown environmental source 2 Met Asn Ser Ser Thr Asn Ala ThrLys Arg Trp Trp Tyr Ile Met Pro 1 5 10 15 Ile Val Phe Ile Thr Tyr SerLeu Ala Tyr Leu Asp Arg Ala Asn Phe 20 25 30 Ser Phe Ala Ser Ala Ala GlyIle Thr Glu Asp Leu Gly Ile Thr Lys 35 40 45 Gly Ile Ser Ser Leu Leu GlyAla Leu Phe Phe Leu Gly Tyr Phe Phe 50 55 60 Phe Gln Ile Pro Gly Ala IleTyr Ala Glu Arg Arg Ser Val Arg Lys 65 70 75 80 Leu Ile Phe Ile Cys LeuIle Leu Trp Gly Ala Cys Ala Ser Leu Asp 85 90 95 Arg Asp Gly Ala Gln TyrSer Ser Ala Gly Trp Arg Ser Val Leu Phe 100 105 110 Ser Ala Val Val GluAla Ala Val Met Pro Ala Met Leu Ile Tyr Ile 115 120 125 Ser Asn Trp PheThr Lys Ser Glu Arg Ser Arg Ala Asn Thr Phe Leu 130 135 140 Ile Leu GlyAsn Pro Val Thr Val Leu Trp Met Ser Val Val Ser Gly 145 150 155 160 TyrLeu Ile Gln Ser Phe Gly Trp Arg Glu Met Phe Ile Ile Glu Gly 165 170 175Val Pro Ala Val Leu Trp Ala Phe Cys Trp Trp Val Leu Val Lys Val 180 185190 Lys Pro Ser Gln Val Asn Trp Leu Ser Glu Asn Glu Lys Ala Ala Leu 195200 205 Gln Ala Gln Leu Glu Ser Glu Gln Gln Gly Ile Lys Ala Val Arg Asn210 215 220 Tyr Gly Glu Ala Phe Arg Ser Arg Asn Val Ile Leu Leu Cys MetGln 225 230 235 240 Tyr Phe Ala Trp Ser Ile Gly Val Tyr Gly Phe Val LeuTrp Leu Pro 245 250 255 Ser Ile Ile Arg Ser Gly Gly Val Asn Met Gly MetVal Glu Val Gly 260 265 270 Trp Leu Ser Ser Val Pro Tyr Leu Ala Ala ThrIle Ala Met Ile Val 275 280 285 Val Ser Trp Ala Ser Asp Lys Met Gln AsnArg Lys Leu Phe Val Trp 290 295 300 Pro Leu Leu Leu Ile Gly Gly Leu AlaPhe Ile Gly Ser Trp Ala Val 305 310 315 320 Gly Ala Asn His Phe Trp AlaSer Tyr Thr Leu Leu Val Ile Ala Asn 325 330 335 Ala Ala Met Tyr Ala ProTyr Gly Pro Phe Phe Ala Ile Ile Pro Glu 340 345 350 Met Leu Pro Arg AsnVal Ala Gly Gly Ala Met Ala Leu Ile Asn Ser 355 360 365 Met Gly Ala LeuGly Ser Phe Phe Gly Ser Trp Phe Val Gly Tyr Leu 370 375 380 Asn Gly ThrThr Gly Ser Pro Ser Ala Ser Tyr Ile Phe Met Gly Val 385 390 395 400 AlaLeu Phe Ala Ser Val Trp Leu Thr Leu Ile Val Lys Pro Ala Asn 405 410 415Asn Gln Lys Leu Pro Ile Gly Ala Arg His Ala 420 425 3 1775 DNA Unknownenvironmental source 3 ggcaatttgc ggtgtttttt ccgcaggacg ttcatcgtccggcctgtatt catcaacggc 60 cctgcgctat tcgcaaagtg gtggtgaaaa taccgctgcgttatttaacg cccaataagc 120 aacaccgagt ttataaccct gaacgacacg gctgcgggcctgtgtagacg cccctacgcc 180 ttaacaccac taaatgactc tacaggtgta tat atg aataca gcc tct gtt tct 234 Met Asn Thr Ala Ser Val Ser 1 5 gtc acc caa agccag gcg atc ccc aaa tta cgc tgg ttg aga ata gtg 282 Val Thr Gln Ser GlnAla Ile Pro Lys Leu Arg Trp Leu Arg Ile Val 10 15 20 ccg cct att ctt attacc tgc att att tcc tat atg gac cgg gtg aac 330 Pro Pro Ile Leu Ile ThrCys Ile Ile Ser Tyr Met Asp Arg Val Asn 25 30 35 atc gcc ttc gcc atg cccggc ggc atg gac gat gaa ctg ggc atc acc 378 Ile Ala Phe Ala Met Pro GlyGly Met Asp Asp Glu Leu Gly Ile Thr 40 45 50 55 gcc tcg atg gcc ggg ttggcc ggc ggt att ttc ttt atc ggt tat ctg 426 Ala Ser Met Ala Gly Leu AlaGly Gly Ile Phe Phe Ile Gly Tyr Leu 60 65 70 ttc ttg cag gta ccc ggc ggcaag ctg gcg gtg tac ggc aac ggc aag 474 Phe Leu Gln Val Pro Gly Gly LysLeu Ala Val Tyr Gly Asn Gly Lys 75 80 85 aaa ttc atc ggt tgg tcg ttg ttggcc tgg gcg gtg att tcc gtg ctg 522 Lys Phe Ile Gly Trp Ser Leu Leu AlaTrp Ala Val Ile Ser Val Leu 90 95 100 acc ggg ctg gtc acg aat cag tatcaa ttg ctg ttc ctg cgc ttc gcc 570 Thr Gly Leu Val Thr Asn Gln Tyr GlnLeu Leu Phe Leu Arg Phe Ala 105 110 115 ctc ggc cgt ttc cga agc ggc atgctg cgg tgg gtg ctg acc atg atc 618 Leu Gly Arg Phe Arg Ser Gly Met LeuArg Trp Val Leu Thr Met Ile 120 125 130 135 agc aac tgg ttc ccg gac aaggaa cgc ggg cgc gcc aac gcc atc gtc 666 Ser Asn Trp Phe Pro Asp Lys GluArg Gly Arg Ala Asn Ala Ile Val 140 145 150 atc atg ttc gtg ccg atc gccggc atc ctt acc gca ccg ctg tcc ggc 714 Ile Met Phe Val Pro Ile Ala GlyIle Leu Thr Ala Pro Leu Ser Gly 155 160 165 tgg atc atc acc gcc tgg gactgg cgc atg ctg ttc ctg gtc gag ggc 762 Trp Ile Ile Thr Ala Trp Asp TrpArg Met Leu Phe Leu Val Glu Gly 170 175 180 gcg ctg tcg ctg gtc gtg atggtg ctg tgg tat ttc acc atc agc aac 810 Ala Leu Ser Leu Val Val Met ValLeu Trp Tyr Phe Thr Ile Ser Asn 185 190 195 cgt cca caa gag gcc aaa aggatt tcg cag gcg gaa aaa gat tat ctg 858 Arg Pro Gln Glu Ala Lys Arg IleSer Gln Ala Glu Lys Asp Tyr Leu 200 205 210 215 atc aaa acg ctg cac gacgaa cag ttg ctg atc aaa ggc aaa acg gtg 906 Ile Lys Thr Leu His Asp GluGln Leu Leu Ile Lys Gly Lys Thr Val 220 225 230 cgc aac gcc tcg ctg cgtcgg gtg ctg ggc gac aaa atc atg tgg aag 954 Arg Asn Ala Ser Leu Arg ArgVal Leu Gly Asp Lys Ile Met Trp Lys 235 240 245 ttc ttc tac cag acc gggata tac ggc tac acc ctg tgg ctg ccg acc 1002 Phe Phe Tyr Gln Thr Gly IleTyr Gly Tyr Thr Leu Trp Leu Pro Thr 250 255 260 att ctc aag ggg ctc accaac ggc aat atg gag cag gtc ggg atg ctg 1050 Ile Leu Lys Gly Leu Thr AsnGly Asn Met Glu Gln Val Gly Met Leu 265 270 275 gct atc ctg ccc tat atcggc gcc atc ttc ggc atg ctg atc att tcc 1098 Ala Ile Leu Pro Tyr Ile GlyAla Ile Phe Gly Met Leu Ile Ile Ser 280 285 290 295 acc ctc tcc gat cgcacc ggc aag cgc aaa gtg ttc gtc gca ctg ccg 1146 Thr Leu Ser Asp Arg ThrGly Lys Arg Lys Val Phe Val Ala Leu Pro 300 305 310 ctg gcc tgc ttt gccatc tgc atg gcg ctg tcg gtg ctg ctg aag gat 1194 Leu Ala Cys Phe Ala IleCys Met Ala Leu Ser Val Leu Leu Lys Asp 315 320 325 cac atc tgg tgg tcgtac gcg gcg ctg gtg ggc tgt ggc gtc ttt acc 1242 His Ile Trp Trp Ser TyrAla Ala Leu Val Gly Cys Gly Val Phe Thr 330 335 340 cag gcc gcc gcc ggggtg ttc tgg acc att ccg ccc aag ctg ttt aac 1290 Gln Ala Ala Ala Gly ValPhe Trp Thr Ile Pro Pro Lys Leu Phe Asn 345 350 355 gcc gaa atg gcc ggcggc gcg cgc ggc gtg atc aat gca ctg ggc aac 1338 Ala Glu Met Ala Gly GlyAla Arg Gly Val Ile Asn Ala Leu Gly Asn 360 365 370 375 ctc ggc ggt ttctgc ggc ccc tat atg gtc ggc gtg ttg atc acc ttg 1386 Leu Gly Gly Phe CysGly Pro Tyr Met Val Gly Val Leu Ile Thr Leu 380 385 390 ttc agc aaa gacgtc ggc gtt tac agc ctc gcg gtg tcg ctg gcc tcc 1434 Phe Ser Lys Asp ValGly Val Tyr Ser Leu Ala Val Ser Leu Ala Ser 395 400 405 gcc tcg gtg ctggcg ttg atg ctg ccg aac aga tgc gac caa aaa gcg 1482 Ala Ser Val Leu AlaLeu Met Leu Pro Asn Arg Cys Asp Gln Lys Ala 410 415 420 ggg gcc gaataatggacta ttggctgggg ctggactgcg gcggcacctt 1531 Gly Ala Glu 425tatcaaagcc ggcctgtatg accggaatgg cgcagaactg ggcatagccc gccgtacgct 1591ggacattgtc gcgccgcaac ccggctgggc ggaacgtgac atgcccgcgc tgtggcagac 1651cgccgccgag gtgatccgcg aattgctggc ccgcaacgac attgccgacg ctgatattca 1711ggccatcggc atctcggcgc agggtaaagg cgcgtttttg ttagacgagc aaggccaacc 1771gttg 1775 4 426 PRT Unknown environmental source 4 Met Asn Thr Ala SerVal Ser Val Thr Gln Ser Gln Ala Ile Pro Lys 1 5 10 15 Leu Arg Trp LeuArg Ile Val Pro Pro Ile Leu Ile Thr Cys Ile Ile 20 25 30 Ser Tyr Met AspArg Val Asn Ile Ala Phe Ala Met Pro Gly Gly Met 35 40 45 Asp Asp Glu LeuGly Ile Thr Ala Ser Met Ala Gly Leu Ala Gly Gly 50 55 60 Ile Phe Phe IleGly Tyr Leu Phe Leu Gln Val Pro Gly Gly Lys Leu 65 70 75 80 Ala Val TyrGly Asn Gly Lys Lys Phe Ile Gly Trp Ser Leu Leu Ala 85 90 95 Trp Ala ValIle Ser Val Leu Thr Gly Leu Val Thr Asn Gln Tyr Gln 100 105 110 Leu LeuPhe Leu Arg Phe Ala Leu Gly Arg Phe Arg Ser Gly Met Leu 115 120 125 ArgTrp Val Leu Thr Met Ile Ser Asn Trp Phe Pro Asp Lys Glu Arg 130 135 140Gly Arg Ala Asn Ala Ile Val Ile Met Phe Val Pro Ile Ala Gly Ile 145 150155 160 Leu Thr Ala Pro Leu Ser Gly Trp Ile Ile Thr Ala Trp Asp Trp Arg165 170 175 Met Leu Phe Leu Val Glu Gly Ala Leu Ser Leu Val Val Met ValLeu 180 185 190 Trp Tyr Phe Thr Ile Ser Asn Arg Pro Gln Glu Ala Lys ArgIle Ser 195 200 205 Gln Ala Glu Lys Asp Tyr Leu Ile Lys Thr Leu His AspGlu Gln Leu 210 215 220 Leu Ile Lys Gly Lys Thr Val Arg Asn Ala Ser LeuArg Arg Val Leu 225 230 235 240 Gly Asp Lys Ile Met Trp Lys Phe Phe TyrGln Thr Gly Ile Tyr Gly 245 250 255 Tyr Thr Leu Trp Leu Pro Thr Ile LeuLys Gly Leu Thr Asn Gly Asn 260 265 270 Met Glu Gln Val Gly Met Leu AlaIle Leu Pro Tyr Ile Gly Ala Ile 275 280 285 Phe Gly Met Leu Ile Ile SerThr Leu Ser Asp Arg Thr Gly Lys Arg 290 295 300 Lys Val Phe Val Ala LeuPro Leu Ala Cys Phe Ala Ile Cys Met Ala 305 310 315 320 Leu Ser Val LeuLeu Lys Asp His Ile Trp Trp Ser Tyr Ala Ala Leu 325 330 335 Val Gly CysGly Val Phe Thr Gln Ala Ala Ala Gly Val Phe Trp Thr 340 345 350 Ile ProPro Lys Leu Phe Asn Ala Glu Met Ala Gly Gly Ala Arg Gly 355 360 365 ValIle Asn Ala Leu Gly Asn Leu Gly Gly Phe Cys Gly Pro Tyr Met 370 375 380Val Gly Val Leu Ile Thr Leu Phe Ser Lys Asp Val Gly Val Tyr Ser 385 390395 400 Leu Ala Val Ser Leu Ala Ser Ala Ser Val Leu Ala Leu Met Leu Pro405 410 415 Asn Arg Cys Asp Gln Lys Ala Gly Ala Glu 420 425 5 1478 DNAKlebsiella oxytoca CDS (73)...(1353) 5 ttgcccgccg ccgctgctca gaccatcgatcttttctatg atgagcgtca cctcactcac 60 agaggcaaac ct atg aat agt tca acgaat gca aca aaa cgc tgg tgg tac 111 Met Asn Ser Ser Thr Asn Ala Thr LysArg Trp Trp Tyr 1 5 10 atc atg cct atc gtg ttt atc acg tat agc ctg gcgtac ctc gac cgc 159 Ile Met Pro Ile Val Phe Ile Thr Tyr Ser Leu Ala TyrLeu Asp Arg 15 20 25 gct aac ttc agc ttc gct tcg gcg gcc gga att act gaagac ctg ggg 207 Ala Asn Phe Ser Phe Ala Ser Ala Ala Gly Ile Thr Glu AspLeu Gly 30 35 40 45 atc acc aaa ggt atc tcc tcc ctt ctg ggg gcg ctg ttcttc ctc ggc 255 Ile Thr Lys Gly Ile Ser Ser Leu Leu Gly Ala Leu Phe PheLeu Gly 50 55 60 tac ttc ttc ttt cag atc ccc ggc gcg att tat gcc gaa cgccgc agc 303 Tyr Phe Phe Phe Gln Ile Pro Gly Ala Ile Tyr Ala Glu Arg ArgSer 65 70 75 gta cgt aaa ctc att ttc atc tgc ctg atc ctg tgg ggt gcc tgcgcc 351 Val Arg Lys Leu Ile Phe Ile Cys Leu Ile Leu Trp Gly Ala Cys Ala80 85 90 tca ctc gac cgg gat ggt gca caa tat tcc cgc gct ggg cgg gcg atc399 Ser Leu Asp Arg Asp Gly Ala Gln Tyr Ser Arg Ala Gly Arg Ala Ile 95100 105 cgc ttt atc ctt ggc gtg gtc gag gcc gca gtc atg ccg gcg atg ctg447 Arg Phe Ile Leu Gly Val Val Glu Ala Ala Val Met Pro Ala Met Leu 110115 120 125 ata tac atc agc aac tgg ttt acc aaa tcc gaa cgc tcg cgc gccaat 495 Ile Tyr Ile Ser Asn Trp Phe Thr Lys Ser Glu Arg Ser Arg Ala Asn130 135 140 acc ttc ctg atc ctc ggc aac ccg gtg acg gtg ctg tgg atg tcggtg 543 Thr Phe Leu Ile Leu Gly Asn Pro Val Thr Val Leu Trp Met Ser Val145 150 155 gtc tcc ggc tac ctg att cag gct ttc ggc tgg cgg gag atg tttatt 591 Val Ser Gly Tyr Leu Ile Gln Ala Phe Gly Trp Arg Glu Met Phe Ile160 165 170 att gaa ggc gtt ccg gcg gtg att tgg gcc ttc tgc tgg tgg gtgctg 639 Ile Glu Gly Val Pro Ala Val Ile Trp Ala Phe Cys Trp Trp Val Leu175 180 185 gta aaa gat aaa ccg tct cag gtc aac tgg ctg gcg gaa agc gaaaag 687 Val Lys Asp Lys Pro Ser Gln Val Asn Trp Leu Ala Glu Ser Glu Lys190 195 200 205 gcc gca ttg cag gag cag ctg gag cgc gaa cag cag ggt atcaaa ccg 735 Ala Ala Leu Gln Glu Gln Leu Glu Arg Glu Gln Gln Gly Ile LysPro 210 215 220 gtg cgc aac tac ggt gag gcc ttc cgc tcg cgt aac gtg gtcctg ctg 783 Val Arg Asn Tyr Gly Glu Ala Phe Arg Ser Arg Asn Val Val LeuLeu 225 230 235 tgc atg caa tat ttc gcc tgg agc atc ggg gtt tac ggt ttcgtg ctg 831 Cys Met Gln Tyr Phe Ala Trp Ser Ile Gly Val Tyr Gly Phe ValLeu 240 245 250 tgg ctg ccg tca att atc cgc agc ggc ggc gag aat atg ggcatg gtc 879 Trp Leu Pro Ser Ile Ile Arg Ser Gly Gly Glu Asn Met Gly MetVal 255 260 265 gag gtc ggc tgg ctc tca tcc gtc ccc tac ctg gcg gca accatc gcc 927 Glu Val Gly Trp Leu Ser Ser Val Pro Tyr Leu Ala Ala Thr IleAla 270 275 280 285 atg atc gtg gtc tcc tgg gcc tcc gat aaa atg cag aaccgc aag cta 975 Met Ile Val Val Ser Trp Ala Ser Asp Lys Met Gln Asn ArgLys Leu 290 295 300 ttc gtc tgg ccg ctg ctg ctg att gcc gcc ttc gcg tttatt ggc tcc 1023 Phe Val Trp Pro Leu Leu Leu Ile Ala Ala Phe Ala Phe IleGly Ser 305 310 315 tgg gcc gtc ggc gct aac cat ttc tgg gtc tct tat accctg ctg gtc 1071 Trp Ala Val Gly Ala Asn His Phe Trp Val Ser Tyr Thr LeuLeu Val 320 325 330 att gcc ggc gcg gcg atg tac gcc ccc tac ggg ccg ttcttc gcc atc 1119 Ile Ala Gly Ala Ala Met Tyr Ala Pro Tyr Gly Pro Phe PheAla Ile 335 340 345 att ccc gag atg ctg ccg cgt aac gtc gcc ggg ggc gccatg gcg ctg 1167 Ile Pro Glu Met Leu Pro Arg Asn Val Ala Gly Gly Ala MetAla Leu 350 355 360 365 att aac agc atg ggc gcg ctg ggt tca ttc ttt ggctca tgg ttt gtc 1215 Ile Asn Ser Met Gly Ala Leu Gly Ser Phe Phe Gly SerTrp Phe Val 370 375 380 ggc tac ctg aac ggc acc acc ggc agc ccg tca gcctcg tac att ttt 1263 Gly Tyr Leu Asn Gly Thr Thr Gly Ser Pro Ser Ala SerTyr Ile Phe 385 390 395 atg gga gtg gcg ctt ttc gtc tcg gta tgg ctt actttg att gtt aag 1311 Met Gly Val Ala Leu Phe Val Ser Val Trp Leu Thr LeuIle Val Lys 400 405 410 cct gct aat aat caa aaa ctt ccg ctc ggc gca cgtcac gcc 1353 Pro Ala Asn Asn Gln Lys Leu Pro Leu Gly Ala Arg His Ala 415420 425 tgaaacatta acgcaacgga gaaccgcatg aagccgtcag tcattctctacaaaacgctt 1413 cccgacgacc tgcaacaagc gtctggaaca acactttacc gtcacgcaggtgaaaaacct 1473 gcgtt 1478 6 427 PRT Klebsiella oxytoca 6 Met Asn SerSer Thr Asn Ala Thr Lys Arg Trp Trp Tyr Ile Met Pro 1 5 10 15 Ile ValPhe Ile Thr Tyr Ser Leu Ala Tyr Leu Asp Arg Ala Asn Phe 20 25 30 Ser PheAla Ser Ala Ala Gly Ile Thr Glu Asp Leu Gly Ile Thr Lys 35 40 45 Gly IleSer Ser Leu Leu Gly Ala Leu Phe Phe Leu Gly Tyr Phe Phe 50 55 60 Phe GlnIle Pro Gly Ala Ile Tyr Ala Glu Arg Arg Ser Val Arg Lys 65 70 75 80 LeuIle Phe Ile Cys Leu Ile Leu Trp Gly Ala Cys Ala Ser Leu Asp 85 90 95 ArgAsp Gly Ala Gln Tyr Ser Arg Ala Gly Arg Ala Ile Arg Phe Ile 100 105 110Leu Gly Val Val Glu Ala Ala Val Met Pro Ala Met Leu Ile Tyr Ile 115 120125 Ser Asn Trp Phe Thr Lys Ser Glu Arg Ser Arg Ala Asn Thr Phe Leu 130135 140 Ile Leu Gly Asn Pro Val Thr Val Leu Trp Met Ser Val Val Ser Gly145 150 155 160 Tyr Leu Ile Gln Ala Phe Gly Trp Arg Glu Met Phe Ile IleGlu Gly 165 170 175 Val Pro Ala Val Ile Trp Ala Phe Cys Trp Trp Val LeuVal Lys Asp 180 185 190 Lys Pro Ser Gln Val Asn Trp Leu Ala Glu Ser GluLys Ala Ala Leu 195 200 205 Gln Glu Gln Leu Glu Arg Glu Gln Gln Gly IleLys Pro Val Arg Asn 210 215 220 Tyr Gly Glu Ala Phe Arg Ser Arg Asn ValVal Leu Leu Cys Met Gln 225 230 235 240 Tyr Phe Ala Trp Ser Ile Gly ValTyr Gly Phe Val Leu Trp Leu Pro 245 250 255 Ser Ile Ile Arg Ser Gly GlyGlu Asn Met Gly Met Val Glu Val Gly 260 265 270 Trp Leu Ser Ser Val ProTyr Leu Ala Ala Thr Ile Ala Met Ile Val 275 280 285 Val Ser Trp Ala SerAsp Lys Met Gln Asn Arg Lys Leu Phe Val Trp 290 295 300 Pro Leu Leu LeuIle Ala Ala Phe Ala Phe Ile Gly Ser Trp Ala Val 305 310 315 320 Gly AlaAsn His Phe Trp Val Ser Tyr Thr Leu Leu Val Ile Ala Gly 325 330 335 AlaAla Met Tyr Ala Pro Tyr Gly Pro Phe Phe Ala Ile Ile Pro Glu 340 345 350Met Leu Pro Arg Asn Val Ala Gly Gly Ala Met Ala Leu Ile Asn Ser 355 360365 Met Gly Ala Leu Gly Ser Phe Phe Gly Ser Trp Phe Val Gly Tyr Leu 370375 380 Asn Gly Thr Thr Gly Ser Pro Ser Ala Ser Tyr Ile Phe Met Gly Val385 390 395 400 Ala Leu Phe Val Ser Val Trp Leu Thr Leu Ile Val Lys ProAla Asn 405 410 415 Asn Gln Lys Leu Pro Leu Gly Ala Arg His Ala 420 4257 1600 DNA Pantoea citrea CDS (214)...(1521) 7 tctgagcttt tcccgaccttatccctgtca gatccctgcc tggttcacga accttgtaac 60 acactttctt aacatgcccttacgtgaccc tgatcccaca ctgtcagtgc aaaaacacgt 120 tttatagctc ctgataagcacagttcgcag cgcgtaactg cacccgcagg gttctgcttt 180 gcgtcaactg acaaaacagaaaggggatat atc atg caa aaa tca cag ccg gga 234 Met Gln Lys Ser Gln ProGly 1 5 acc cgc tgg ttt cgg att att gtg ccg atc ctg ata gcc tgc atc atg282 Thr Arg Trp Phe Arg Ile Ile Val Pro Ile Leu Ile Ala Cys Ile Met 1015 20 tcg ttt atg gat cgg gta aat atc agt ttc gca ttg ccg ggc ggt atg330 Ser Phe Met Asp Arg Val Asn Ile Ser Phe Ala Leu Pro Gly Gly Met 2530 35 gag cag gat ctg ctg atg tcc agc cag atg gcc ggg gta gtt agc ggt378 Glu Gln Asp Leu Leu Met Ser Ser Gln Met Ala Gly Val Val Ser Gly 4045 50 55 att ttc ttt att ggt tat ctg ttt ttg cag gtt cct ggt ggg cat atc426 Ile Phe Phe Ile Gly Tyr Leu Phe Leu Gln Val Pro Gly Gly His Ile 6065 70 gca gta cgt ggc agt ggt aaa cgt ttt att gcc tgg tcg ctt gtt gcc474 Ala Val Arg Gly Ser Gly Lys Arg Phe Ile Ala Trp Ser Leu Val Ala 7580 85 tgg gcc gtt gtt tct gtc gct acc ggg ttt gtg act cat cag tac cag522 Trp Ala Val Val Ser Val Ala Thr Gly Phe Val Thr His Gln Tyr Gln 9095 100 ctg ttg att tta cgt ttt gca ctg ggg gtc tct gaa ggt ggg atg ttg570 Leu Leu Ile Leu Arg Phe Ala Leu Gly Val Ser Glu Gly Gly Met Leu 105110 115 ccg gta gtt ctg aca atg gtc agc aac tgg ttt cct gaa aaa gag ctg618 Pro Val Val Leu Thr Met Val Ser Asn Trp Phe Pro Glu Lys Glu Leu 120125 130 135 ggg cgt gct aat gca ttt gtc atg atg ttc gcc ccg ctt ggc ggaatg 666 Gly Arg Ala Asn Ala Phe Val Met Met Phe Ala Pro Leu Gly Gly Met140 145 150 att acc gcc cct gtc tcc gga tgg att att gca ctg cta gac tggcgc 714 Ile Thr Ala Pro Val Ser Gly Trp Ile Ile Ala Leu Leu Asp Trp Arg155 160 165 tgg tta ttt att atc gaa gga tta ctg tcg gta gtg gtt ctg gcagtc 762 Trp Leu Phe Ile Ile Glu Gly Leu Leu Ser Val Val Val Leu Ala Val170 175 180 tgg tgg ctg atg gtc agt gac cgc cct gaa gat gcc cgt tgg ctgccg 810 Trp Trp Leu Met Val Ser Asp Arg Pro Glu Asp Ala Arg Trp Leu Pro185 190 195 gca gca gaa cgg gaa tat ctg ctg cgc gaa atg gcc cgt gac aaggcc 858 Ala Ala Glu Arg Glu Tyr Leu Leu Arg Glu Met Ala Arg Asp Lys Ala200 205 210 215 gag cgg agc aaa ctc cct ccg atc agt cat gct ccc ctg caagag gtt 906 Glu Arg Ser Lys Leu Pro Pro Ile Ser His Ala Pro Leu Gln GluVal 220 225 230 ttc cat aac ccg ggc ctg atg aag tta gtg att ctg aac tttttc tat 954 Phe His Asn Pro Gly Leu Met Lys Leu Val Ile Leu Asn Phe PheTyr 235 240 245 cag aca ggt gat tac gga tac act ctg tgg ctg ccg act attatc aaa 1002 Gln Thr Gly Asp Tyr Gly Tyr Thr Leu Trp Leu Pro Thr Ile IleLys 250 255 260 aac ctg acc gga gct agt att ggt aac gtc ggt ttg ctg acagtg cta 1050 Asn Leu Thr Gly Ala Ser Ile Gly Asn Val Gly Leu Leu Thr ValLeu 265 270 275 cct ttt atc gcg acg tta tca ggg att tat gtc gtc tct tacctg agc 1098 Pro Phe Ile Ala Thr Leu Ser Gly Ile Tyr Val Val Ser Tyr LeuSer 280 285 290 295 gat aaa acc ggc aaa cgt cgg caa tgg gtg atg att tctctg ttc tgt 1146 Asp Lys Thr Gly Lys Arg Arg Gln Trp Val Met Ile Ser LeuPhe Cys 300 305 310 ttt gcg gcc tgc ctg ttg gcc tca gtc ctg tta cgt gaattt gtg ctg 1194 Phe Ala Ala Cys Leu Leu Ala Ser Val Leu Leu Arg Glu PheVal Leu 315 320 325 gct gct tat ctg gct ctg gtg gct tgc ggc ttt ttc ctgaaa gca gcc 1242 Ala Ala Tyr Leu Ala Leu Val Ala Cys Gly Phe Phe Leu LysAla Ala 330 335 340 acc agc ccg ttc tgg agt att ccg gga cgt att gca ccgccg gaa gca 1290 Thr Ser Pro Phe Trp Ser Ile Pro Gly Arg Ile Ala Pro ProGlu Ala 345 350 355 gcc ggt agt gcc cgt ggt gta att aac gga ctg ggg aatctg ggc ggt 1338 Ala Gly Ser Ala Arg Gly Val Ile Asn Gly Leu Gly Asn LeuGly Gly 360 365 370 375 ttc tgc ggc ccc tgg ctg gtc gga tta atg atc tacctg tac gga cag 1386 Phe Cys Gly Pro Trp Leu Val Gly Leu Met Ile Tyr LeuTyr Gly Gln 380 385 390 aat gca gcc gtt gtt act ctg gca ggc tct ctg atcatt gcc ggg att 1434 Asn Ala Ala Val Val Thr Leu Ala Gly Ser Leu Ile IleAla Gly Ile 395 400 405 att gcg gca tta ctg cca acg cag tgt gat ctg cgcccg gca gag gca 1482 Ile Ala Ala Leu Leu Pro Thr Gln Cys Asp Leu Arg ProAla Glu Ala 410 415 420 cgg cag cag aat ttc acc cca cgt att cat gat gccaaa taatactgtc 1531 Arg Gln Gln Asn Phe Thr Pro Arg Ile His Asp Ala Lys425 430 435 acccggtaac gctgttgccg ggtgcagcct tcacctttca gggcgtatttttctgataac 1591 cccgtgtaa 1600 8 436 PRT Pantoea citrea 8 Met Gln LysSer Gln Pro Gly Thr Arg Trp Phe Arg Ile Ile Val Pro 1 5 10 15 Ile LeuIle Ala Cys Ile Met Ser Phe Met Asp Arg Val Asn Ile Ser 20 25 30 Phe AlaLeu Pro Gly Gly Met Glu Gln Asp Leu Leu Met Ser Ser Gln 35 40 45 Met AlaGly Val Val Ser Gly Ile Phe Phe Ile Gly Tyr Leu Phe Leu 50 55 60 Gln ValPro Gly Gly His Ile Ala Val Arg Gly Ser Gly Lys Arg Phe 65 70 75 80 IleAla Trp Ser Leu Val Ala Trp Ala Val Val Ser Val Ala Thr Gly 85 90 95 PheVal Thr His Gln Tyr Gln Leu Leu Ile Leu Arg Phe Ala Leu Gly 100 105 110Val Ser Glu Gly Gly Met Leu Pro Val Val Leu Thr Met Val Ser Asn 115 120125 Trp Phe Pro Glu Lys Glu Leu Gly Arg Ala Asn Ala Phe Val Met Met 130135 140 Phe Ala Pro Leu Gly Gly Met Ile Thr Ala Pro Val Ser Gly Trp Ile145 150 155 160 Ile Ala Leu Leu Asp Trp Arg Trp Leu Phe Ile Ile Glu GlyLeu Leu 165 170 175 Ser Val Val Val Leu Ala Val Trp Trp Leu Met Val SerAsp Arg Pro 180 185 190 Glu Asp Ala Arg Trp Leu Pro Ala Ala Glu Arg GluTyr Leu Leu Arg 195 200 205 Glu Met Ala Arg Asp Lys Ala Glu Arg Ser LysLeu Pro Pro Ile Ser 210 215 220 His Ala Pro Leu Gln Glu Val Phe His AsnPro Gly Leu Met Lys Leu 225 230 235 240 Val Ile Leu Asn Phe Phe Tyr GlnThr Gly Asp Tyr Gly Tyr Thr Leu 245 250 255 Trp Leu Pro Thr Ile Ile LysAsn Leu Thr Gly Ala Ser Ile Gly Asn 260 265 270 Val Gly Leu Leu Thr ValLeu Pro Phe Ile Ala Thr Leu Ser Gly Ile 275 280 285 Tyr Val Val Ser TyrLeu Ser Asp Lys Thr Gly Lys Arg Arg Gln Trp 290 295 300 Val Met Ile SerLeu Phe Cys Phe Ala Ala Cys Leu Leu Ala Ser Val 305 310 315 320 Leu LeuArg Glu Phe Val Leu Ala Ala Tyr Leu Ala Leu Val Ala Cys 325 330 335 GlyPhe Phe Leu Lys Ala Ala Thr Ser Pro Phe Trp Ser Ile Pro Gly 340 345 350Arg Ile Ala Pro Pro Glu Ala Ala Gly Ser Ala Arg Gly Val Ile Asn 355 360365 Gly Leu Gly Asn Leu Gly Gly Phe Cys Gly Pro Trp Leu Val Gly Leu 370375 380 Met Ile Tyr Leu Tyr Gly Gln Asn Ala Ala Val Val Thr Leu Ala Gly385 390 395 400 Ser Leu Ile Ile Ala Gly Ile Ile Ala Ala Leu Leu Pro ThrGln Cys 405 410 415 Asp Leu Arg Pro Ala Glu Ala Arg Gln Gln Asn Phe ThrPro Arg Ile 420 425 430 His Asp Ala Lys 435 9 1500 DNA Pantoea citreaCDS (154)...(1440) 9 tcagctcagg cctcggattt cgttaacggt catgtcttatttgtcgacgg cggaatgctg 60 gcctcagtat aaaatacagg ggcagacgga atcagagtttgccctgaaga tatcttactg 120 gttgcccctt cggcacacac aggatgttcc ccc atg aataca agc aga aaa ctg 174 Met Asn Thr Ser Arg Lys Leu 1 5 ccg gtg aaa cgctgg tgg tat tta atg ccg gtg att ttt att act tac 222 Pro Val Lys Arg TrpTrp Tyr Leu Met Pro Val Ile Phe Ile Thr Tyr 10 15 20 agc ctg gca tat ctggat cgg gcc aac tac ggc ttt gct gct gcc tct 270 Ser Leu Ala Tyr Leu AspArg Ala Asn Tyr Gly Phe Ala Ala Ala Ser 25 30 35 ggg att gaa gca gat cttgga att agc cgt ggc acc tcc tct ctg att 318 Gly Ile Glu Ala Asp Leu GlyIle Ser Arg Gly Thr Ser Ser Leu Ile 40 45 50 55 gga gca ctg ttc ttt ctcggc tac ttc att ttt cag gtg ccc ggg gca 366 Gly Ala Leu Phe Phe Leu GlyTyr Phe Ile Phe Gln Val Pro Gly Ala 60 65 70 att tat gca gtg aaa cgc agtgtc cgt aaa ctg gtg ttt acc agc ctg 414 Ile Tyr Ala Val Lys Arg Ser ValArg Lys Leu Val Phe Thr Ser Leu 75 80 85 ctg ttg tgg gga ttt tgt gcc gctgcg acc gga ctt atc agc aat att 462 Leu Leu Trp Gly Phe Cys Ala Ala AlaThr Gly Leu Ile Ser Asn Ile 90 95 100 ccg gct ctg atg gtg atc cgc tttgtt ctg ggt gtt gtt gaa gcc gca 510 Pro Ala Leu Met Val Ile Arg Phe ValLeu Gly Val Val Glu Ala Ala 105 110 115 gtg atg cca gcg atg ctg att tacatc agc aac tgg ttc acc cgt cag 558 Val Met Pro Ala Met Leu Ile Tyr IleSer Asn Trp Phe Thr Arg Gln 120 125 130 135 gaa cgt tca cgg gct aat accttt ctg gta tta ggt aac ccg gtc acg 606 Glu Arg Ser Arg Ala Asn Thr PheLeu Val Leu Gly Asn Pro Val Thr 140 145 150 gtg tta tgg atg tct att gtttcc gga tat ctg atc aat gct ttt ggc 654 Val Leu Trp Met Ser Ile Val SerGly Tyr Leu Ile Asn Ala Phe Gly 155 160 165 tgg cgg gaa atg ttt att ttcgag ggt gtg cct gcc tta atc tgg gcc 702 Trp Arg Glu Met Phe Ile Phe GluGly Val Pro Ala Leu Ile Trp Ala 170 175 180 atc ttc tgg tgg ttt att gtccgg gac aaa ccg gag cag gtg agc tgg 750 Ile Phe Trp Trp Phe Ile Val ArgAsp Lys Pro Glu Gln Val Ser Trp 185 190 195 ctg aca gaa aca gaa aag cagcaa ctg gcc agt gca atg gct gaa gag 798 Leu Thr Glu Thr Glu Lys Gln GlnLeu Ala Ser Ala Met Ala Glu Glu 200 205 210 215 cag cag gca ata cca ccgatg cgc aat gtg ccg cag gcc ctg cgt tcc 846 Gln Gln Ala Ile Pro Pro MetArg Asn Val Pro Gln Ala Leu Arg Ser 220 225 230 cgc aat gtg gtg gta ctgtgc ctg tta cac gct ctg tgg agc atc gga 894 Arg Asn Val Val Val Leu CysLeu Leu His Ala Leu Trp Ser Ile Gly 235 240 245 gtg tat ggt ttt atg atgtgg atg cca tcg ata ctg cgt agc gct gca 942 Val Tyr Gly Phe Met Met TrpMet Pro Ser Ile Leu Arg Ser Ala Ala 250 255 260 tca atg gac att gtc cgggta ggc tgg ctg gcc gca gtt ccg tat ctg 990 Ser Met Asp Ile Val Arg ValGly Trp Leu Ala Ala Val Pro Tyr Leu 265 270 275 gcc gcg att att act atgctg gtg att tca tgg ctg tca gat aaa acc 1038 Ala Ala Ile Ile Thr Met LeuVal Ile Ser Trp Leu Ser Asp Lys Thr 280 285 290 295 ggg ctg cgt cgg cttttt atc tgg cca tta ttg ctg att gcg tca gtt 1086 Gly Leu Arg Arg Leu PheIle Trp Pro Leu Leu Leu Ile Ala Ser Val 300 305 310 act ttt ttt ggg tcctgg tta ctt ggg agc tac tca ttc tgg ttt tcc 1134 Thr Phe Phe Gly Ser TrpLeu Leu Gly Ser Tyr Ser Phe Trp Phe Ser 315 320 325 tat ggc ttg ctg gtactg gct gct gct tgt atg tat gcc ccg tat gga 1182 Tyr Gly Leu Leu Val LeuAla Ala Ala Cys Met Tyr Ala Pro Tyr Gly 330 335 340 ccg ttt ttt gcg ttgatt cct gaa ttg ctg cca aaa aat gtg gcg ggg 1230 Pro Phe Phe Ala Leu IlePro Glu Leu Leu Pro Lys Asn Val Ala Gly 345 350 355 att tct atc ggg ttaatt aac tgt tgc ggg gcg ctg gga gct ttt gcc 1278 Ile Ser Ile Gly Leu IleAsn Cys Cys Gly Ala Leu Gly Ala Phe Ala 360 365 370 375 gga gcc tgg ctggtg ggc tat ctt aat ggt ctg acc ggt ggt ccg ggg 1326 Gly Ala Trp Leu ValGly Tyr Leu Asn Gly Leu Thr Gly Gly Pro Gly 380 385 390 gct tct tac actttt atg gcc att gca ttg ctg gtt tct gta ggg ttg 1374 Ala Ser Tyr Thr PheMet Ala Ile Ala Leu Leu Val Ser Val Gly Leu 395 400 405 gtg ttt ttc ctgaaa gtc cct tca ggg aat ttg gtc act cgt cgg ttg 1422 Val Phe Phe Leu LysVal Pro Ser Gly Asn Leu Val Thr Arg Arg Leu 410 415 420 ctg aaa ggt gatgca aag taaaaggaat agcgatgaaa cggaacagga 1470 Leu Lys Gly Asp Ala Lys425 tgtctttgca ggatattgcg gacctcgccg 1500 10 429 PRT Pantoea citrea 10Met Asn Thr Ser Arg Lys Leu Pro Val Lys Arg Trp Trp Tyr Leu Met 1 5 1015 Pro Val Ile Phe Ile Thr Tyr Ser Leu Ala Tyr Leu Asp Arg Ala Asn 20 2530 Tyr Gly Phe Ala Ala Ala Ser Gly Ile Glu Ala Asp Leu Gly Ile Ser 35 4045 Arg Gly Thr Ser Ser Leu Ile Gly Ala Leu Phe Phe Leu Gly Tyr Phe 50 5560 Ile Phe Gln Val Pro Gly Ala Ile Tyr Ala Val Lys Arg Ser Val Arg 65 7075 80 Lys Leu Val Phe Thr Ser Leu Leu Leu Trp Gly Phe Cys Ala Ala Ala 8590 95 Thr Gly Leu Ile Ser Asn Ile Pro Ala Leu Met Val Ile Arg Phe Val100 105 110 Leu Gly Val Val Glu Ala Ala Val Met Pro Ala Met Leu Ile TyrIle 115 120 125 Ser Asn Trp Phe Thr Arg Gln Glu Arg Ser Arg Ala Asn ThrPhe Leu 130 135 140 Val Leu Gly Asn Pro Val Thr Val Leu Trp Met Ser IleVal Ser Gly 145 150 155 160 Tyr Leu Ile Asn Ala Phe Gly Trp Arg Glu MetPhe Ile Phe Glu Gly 165 170 175 Val Pro Ala Leu Ile Trp Ala Ile Phe TrpTrp Phe Ile Val Arg Asp 180 185 190 Lys Pro Glu Gln Val Ser Trp Leu ThrGlu Thr Glu Lys Gln Gln Leu 195 200 205 Ala Ser Ala Met Ala Glu Glu GlnGln Ala Ile Pro Pro Met Arg Asn 210 215 220 Val Pro Gln Ala Leu Arg SerArg Asn Val Val Val Leu Cys Leu Leu 225 230 235 240 His Ala Leu Trp SerIle Gly Val Tyr Gly Phe Met Met Trp Met Pro 245 250 255 Ser Ile Leu ArgSer Ala Ala Ser Met Asp Ile Val Arg Val Gly Trp 260 265 270 Leu Ala AlaVal Pro Tyr Leu Ala Ala Ile Ile Thr Met Leu Val Ile 275 280 285 Ser TrpLeu Ser Asp Lys Thr Gly Leu Arg Arg Leu Phe Ile Trp Pro 290 295 300 LeuLeu Leu Ile Ala Ser Val Thr Phe Phe Gly Ser Trp Leu Leu Gly 305 310 315320 Ser Tyr Ser Phe Trp Phe Ser Tyr Gly Leu Leu Val Leu Ala Ala Ala 325330 335 Cys Met Tyr Ala Pro Tyr Gly Pro Phe Phe Ala Leu Ile Pro Glu Leu340 345 350 Leu Pro Lys Asn Val Ala Gly Ile Ser Ile Gly Leu Ile Asn CysCys 355 360 365 Gly Ala Leu Gly Ala Phe Ala Gly Ala Trp Leu Val Gly TyrLeu Asn 370 375 380 Gly Leu Thr Gly Gly Pro Gly Ala Ser Tyr Thr Phe MetAla Ile Ala 385 390 395 400 Leu Leu Val Ser Val Gly Leu Val Phe Phe LeuLys Val Pro Ser Gly 405 410 415 Asn Leu Val Thr Arg Arg Leu Leu Lys GlyAsp Ala Lys 420 425 11 1500 DNA Klebsiella oxytoca CDS (70)...(1386) 11ctaaaacaag cacaataata ataatcacct tcatcaccag aatattttta atattacgag 60actataaag atg aat ata acc tct aac tct aca acc aaa gat ata ccg cgc 111Met Asn Ile Thr Ser Asn Ser Thr Thr Lys Asp Ile Pro Arg 1 5 10 cag cgctgg tta aga atc att ccg cct ata ctg atc act tgt att att 159 Gln Arg TrpLeu Arg Ile Ile Pro Pro Ile Leu Ile Thr Cys Ile Ile 15 20 25 30 tct tatatg gac cgg gtc aat att gcc ttt gcg atg ccc gga ggt atg 207 Ser Tyr MetAsp Arg Val Asn Ile Ala Phe Ala Met Pro Gly Gly Met 35 40 45 gat gcc gactta ggt att tcc gcc acc atg gcg ggg ctg gcg ggc ggt 255 Asp Ala Asp LeuGly Ile Ser Ala Thr Met Ala Gly Leu Ala Gly Gly 50 55 60 att ttc ttt atcggt tat cta ttt tta cag gtt ccc ggc ggg aaa att 303 Ile Phe Phe Ile GlyTyr Leu Phe Leu Gln Val Pro Gly Gly Lys Ile 65 70 75 gcc gtt cac ggt agcggt aag aaa ttt atc ggc tgg tcg ctg gtc gcc 351 Ala Val His Gly Ser GlyLys Lys Phe Ile Gly Trp Ser Leu Val Ala 80 85 90 tgg gcg gtc atc tcc gtgctg acg ggg tta att acc aat cag tac cag 399 Trp Ala Val Ile Ser Val LeuThr Gly Leu Ile Thr Asn Gln Tyr Gln 95 100 105 110 ctg ctg gcc ctg cgcttc tta ctg ggc gtg gcg gaa ggc ggt atg ctg 447 Leu Leu Ala Leu Arg PheLeu Leu Gly Val Ala Glu Gly Gly Met Leu 115 120 125 ccg gtc gtt ctc acgatg atc agt aac tgg ttc ccc gac gct gaa cgc 495 Pro Val Val Leu Thr MetIle Ser Asn Trp Phe Pro Asp Ala Glu Arg 130 135 140 ggt cgc gcc aac gcgatt gtc att atg ttt gtg ccg att gcc ggg att 543 Gly Arg Ala Asn Ala IleVal Ile Met Phe Val Pro Ile Ala Gly Ile 145 150 155 atc acc gcc cca ctctca ggc tgg att atc acg gtt ctc gac tgg cgc 591 Ile Thr Ala Pro Leu SerGly Trp Ile Ile Thr Val Leu Asp Trp Arg 160 165 170 tgg ctg ttt att atcgaa ggt ttg ctc tcg ctg gtt gtt ctg gtt ctg 639 Trp Leu Phe Ile Ile GluGly Leu Leu Ser Leu Val Val Leu Val Leu 175 180 185 190 tgg gca tac accatc tat gac cgt ccg cag gaa gcg cgc tgg att tcc 687 Trp Ala Tyr Thr IleTyr Asp Arg Pro Gln Glu Ala Arg Trp Ile Ser 195 200 205 gaa gca gag aagcgc tat ctg gtc gag acg ctg gcc gcg gag caa aaa 735 Glu Ala Glu Lys ArgTyr Leu Val Glu Thr Leu Ala Ala Glu Gln Lys 210 215 220 gcc att gcc ggcacc gag gtg aaa aac gcc tct ctg agc gcc gtt ctc 783 Ala Ile Ala Gly ThrGlu Val Lys Asn Ala Ser Leu Ser Ala Val Leu 225 230 235 tcc gac aaa accatg tgg cag ctt atc gcc ctg aac ttc ttc tac cag 831 Ser Asp Lys Thr MetTrp Gln Leu Ile Ala Leu Asn Phe Phe Tyr Gln 240 245 250 acc ggc att tacggc tac acc ctg tgg cta ccc acc att ctg aaa gaa 879 Thr Gly Ile Tyr GlyTyr Thr Leu Trp Leu Pro Thr Ile Leu Lys Glu 255 260 265 270 ttg acc catagc agc atg ggg cag gtc ggc atg ctt gcc att ctg ccg 927 Leu Thr His SerSer Met Gly Gln Val Gly Met Leu Ala Ile Leu Pro 275 280 285 tac gtc ggcgcc att gct ggg atg ttc ctg ttt tcc tcc ctt tca gac 975 Tyr Val Gly AlaIle Ala Gly Met Phe Leu Phe Ser Ser Leu Ser Asp 290 295 300 cga acc ggtaaa cgc aag ctg ttc gtc tgc ctg ccg ctg att ggc ttc 1023 Arg Thr Gly LysArg Lys Leu Phe Val Cys Leu Pro Leu Ile Gly Phe 305 310 315 gct ctg tgcatg ttc ctg tcg gtg gcg ctg aaa aac caa att tgg ctc 1071 Ala Leu Cys MetPhe Leu Ser Val Ala Leu Lys Asn Gln Ile Trp Leu 320 325 330 tcc tat gccgcg ctg gtc ggc tgc gga ttc ttc ctg caa tcg gcg gct 1119 Ser Tyr Ala AlaLeu Val Gly Cys Gly Phe Phe Leu Gln Ser Ala Ala 335 340 345 350 ggc gtgttc tgg acc atc ccg gca cgt ctg ttc agc gcg gaa atg gcg 1167 Gly Val PheTrp Thr Ile Pro Ala Arg Leu Phe Ser Ala Glu Met Ala 355 360 365 ggc ggcgcg cgc ggg gtt atc aac gcg ctt ggc aac ctc ggc gga ttt 1215 Gly Gly AlaArg Gly Val Ile Asn Ala Leu Gly Asn Leu Gly Gly Phe 370 375 380 tgt ggccct tat gcg gtc ggg gtg ctg atc acg ttg tac agc aaa gac 1263 Cys Gly ProTyr Ala Val Gly Val Leu Ile Thr Leu Tyr Ser Lys Asp 385 390 395 gct ggcgtc tat tgc ctg gcg atc tcc ctg gcg ctg gcc gcg ctg atg 1311 Ala Gly ValTyr Cys Leu Ala Ile Ser Leu Ala Leu Ala Ala Leu Met 400 405 410 gcg ctgctg ctg ccg gcg aaa tgc gat gcc ggt gct gcg ccg gta aag 1359 Ala Leu LeuLeu Pro Ala Lys Cys Asp Ala Gly Ala Ala Pro Val Lys 415 420 425 430 acgata aat cca cat aaa cgc act gcg taaactcgag cccggcggcg 1406 Thr Ile AsnPro His Lys Arg Thr Ala 435 ctgcgcctgc cgggcctgcg aaatatgccg ggttcacccggtaacaatga gatgcgaaag 1466 atgagcaaga aacaggcctt ctggctgggt attg 1500 12439 PRT Klebsiella oxytoca 12 Met Asn Ile Thr Ser Asn Ser Thr Thr LysAsp Ile Pro Arg Gln Arg 1 5 10 15 Trp Leu Arg Ile Ile Pro Pro Ile LeuIle Thr Cys Ile Ile Ser Tyr 20 25 30 Met Asp Arg Val Asn Ile Ala Phe AlaMet Pro Gly Gly Met Asp Ala 35 40 45 Asp Leu Gly Ile Ser Ala Thr Met AlaGly Leu Ala Gly Gly Ile Phe 50 55 60 Phe Ile Gly Tyr Leu Phe Leu Gln ValPro Gly Gly Lys Ile Ala Val 65 70 75 80 His Gly Ser Gly Lys Lys Phe IleGly Trp Ser Leu Val Ala Trp Ala 85 90 95 Val Ile Ser Val Leu Thr Gly LeuIle Thr Asn Gln Tyr Gln Leu Leu 100 105 110 Ala Leu Arg Phe Leu Leu GlyVal Ala Glu Gly Gly Met Leu Pro Val 115 120 125 Val Leu Thr Met Ile SerAsn Trp Phe Pro Asp Ala Glu Arg Gly Arg 130 135 140 Ala Asn Ala Ile ValIle Met Phe Val Pro Ile Ala Gly Ile Ile Thr 145 150 155 160 Ala Pro LeuSer Gly Trp Ile Ile Thr Val Leu Asp Trp Arg Trp Leu 165 170 175 Phe IleIle Glu Gly Leu Leu Ser Leu Val Val Leu Val Leu Trp Ala 180 185 190 TyrThr Ile Tyr Asp Arg Pro Gln Glu Ala Arg Trp Ile Ser Glu Ala 195 200 205Glu Lys Arg Tyr Leu Val Glu Thr Leu Ala Ala Glu Gln Lys Ala Ile 210 215220 Ala Gly Thr Glu Val Lys Asn Ala Ser Leu Ser Ala Val Leu Ser Asp 225230 235 240 Lys Thr Met Trp Gln Leu Ile Ala Leu Asn Phe Phe Tyr Gln ThrGly 245 250 255 Ile Tyr Gly Tyr Thr Leu Trp Leu Pro Thr Ile Leu Lys GluLeu Thr 260 265 270 His Ser Ser Met Gly Gln Val Gly Met Leu Ala Ile LeuPro Tyr Val 275 280 285 Gly Ala Ile Ala Gly Met Phe Leu Phe Ser Ser LeuSer Asp Arg Thr 290 295 300 Gly Lys Arg Lys Leu Phe Val Cys Leu Pro LeuIle Gly Phe Ala Leu 305 310 315 320 Cys Met Phe Leu Ser Val Ala Leu LysAsn Gln Ile Trp Leu Ser Tyr 325 330 335 Ala Ala Leu Val Gly Cys Gly PhePhe Leu Gln Ser Ala Ala Gly Val 340 345 350 Phe Trp Thr Ile Pro Ala ArgLeu Phe Ser Ala Glu Met Ala Gly Gly 355 360 365 Ala Arg Gly Val Ile AsnAla Leu Gly Asn Leu Gly Gly Phe Cys Gly 370 375 380 Pro Tyr Ala Val GlyVal Leu Ile Thr Leu Tyr Ser Lys Asp Ala Gly 385 390 395 400 Val Tyr CysLeu Ala Ile Ser Leu Ala Leu Ala Ala Leu Met Ala Leu 405 410 415 Leu LeuPro Ala Lys Cys Asp Ala Gly Ala Ala Pro Val Lys Thr Ile 420 425 430 AsnPro His Lys Arg Thr Ala 435 13 3153 DNA Unknown environmental source 13catgcctgca ggtcgactct agaggatctc gccgcgcctc aggtcgaggg atacactcgt 60cagcgctttc gtgccgccga actccttcga aacggcacga aactccagaa gtttgtccgt 120atccaccccg ctcctcccaa agctttatga ggctatagga tattgatatg gtatcgataa 180cactcctgtc aagaggcggt tttcacgcca ggcgggaggg caaaatagga ctggacaatt 240ccttcaagcg ggatatgtta tcgataacaa atcatcttcc ggaggagagc c atg agc 297 MetSer 1 aag atc gat gtg ttg cag gtc ggt ccc tac cct gca tgg gac gag gag345 Lys Ile Asp Val Leu Gln Val Gly Pro Tyr Pro Ala Trp Asp Glu Glu 5 1015 cgc ctg aac gcg acc ttc acg atg cac cgc tat ttc gag gcg gcc gac 393Arg Leu Asn Ala Thr Phe Thr Met His Arg Tyr Phe Glu Ala Ala Asp 20 25 30aag gcg gcg ttt ctg gcc gag cac ggc ggc acg atc cgc ggc atc gcc 441 LysAla Ala Phe Leu Ala Glu His Gly Gly Thr Ile Arg Gly Ile Ala 35 40 45 50acg cgc ggc gag ctt ggt gcc aac cgg gcg atg atc gag gcg ctg ccg 489 ThrArg Gly Glu Leu Gly Ala Asn Arg Ala Met Ile Glu Ala Leu Pro 55 60 65 aagctg gaa gtg atc tcg gtc tac ggc gtc ggc ttc gat gcg gtg gac 537 Lys LeuGlu Val Ile Ser Val Tyr Gly Val Gly Phe Asp Ala Val Asp 70 75 80 ctt tcggcg gcc cgc gag cgc ggc atc cgc gtc acc aac acg ccc gac 585 Leu Ser AlaAla Arg Glu Arg Gly Ile Arg Val Thr Asn Thr Pro Asp 85 90 95 gtg ctc accaag gac gtg gcc gat ctc ggc atc gcc atg atg ctg gcg 633 Val Leu Thr LysAsp Val Ala Asp Leu Gly Ile Ala Met Met Leu Ala 100 105 110 cag gcg cgcggc gtc atc ggc gga gag gcc tgg gtg aag agc ggc gat 681 Gln Ala Arg GlyVal Ile Gly Gly Glu Ala Trp Val Lys Ser Gly Asp 115 120 125 130 tgg gcaagc aag ggt ctc tat ccg ctg aag cgc cgc gta cat ggc atg 729 Trp Ala SerLys Gly Leu Tyr Pro Leu Lys Arg Arg Val His Gly Met 135 140 145 cgc gccggg gtg ctc ggc ctc ggc cgc atc ggc tac gag gtg gcc aag 777 Arg Ala GlyVal Leu Gly Leu Gly Arg Ile Gly Tyr Glu Val Ala Lys 150 155 160 cgc cttgcc ggc ttc gac atg gac atc gcc tac agc gac acc ggc ccg 825 Arg Leu AlaGly Phe Asp Met Asp Ile Ala Tyr Ser Asp Thr Gly Pro 165 170 175 aag gatttc gcc agg gac tgg acc ttc gtc gcc gat ccg gcg gag ctg 873 Lys Asp PheAla Arg Asp Trp Thr Phe Val Ala Asp Pro Ala Glu Leu 180 185 190 gcc gcccgc tcc gac ttc ctc ttc gtc acg ctc gcc gcc tcc gcc gag 921 Ala Ala ArgSer Asp Phe Leu Phe Val Thr Leu Ala Ala Ser Ala Glu 195 200 205 210 acgcgc cac atc gtc ggc cgc aag gtc atc gag gcg ctc ggc cct gag 969 Thr ArgHis Ile Val Gly Arg Lys Val Ile Glu Ala Leu Gly Pro Glu 215 220 225 ggcatg ctg atc aac atc tcg cgc gct tcc aac atc gat gaa agc gcc 1017 Gly MetLeu Ile Asn Ile Ser Arg Ala Ser Asn Ile Asp Glu Ser Ala 230 235 240 cttctc gac gcg ctg gag acg aag gcg ctc ggc tcg gcc gcg ctc gac 1065 Leu LeuAsp Ala Leu Glu Thr Lys Ala Leu Gly Ser Ala Ala Leu Asp 245 250 255 gtcttc gag ggc gag ccg aac ctc aat ccg cgt ttc ctt gcc ctc gac 1113 Val PheGlu Gly Glu Pro Asn Leu Asn Pro Arg Phe Leu Ala Leu Asp 260 265 270 aacgtc ctc ttg cag ccg cac atg gcc tcc ggc acg atc gag acc cgc 1161 Asn ValLeu Leu Gln Pro His Met Ala Ser Gly Thr Ile Glu Thr Arg 275 280 285 290aag gcc atg ggc cag ctc gtc ttc gac aac ctg tcg gcc cat ttc gac 1209 LysAla Met Gly Gln Leu Val Phe Asp Asn Leu Ser Ala His Phe Asp 295 300 305ggc cgg ccg ctg ccg acc ccg gtt ctg taaggagaga ggtcc atg aag gcg 1260Gly Arg Pro Leu Pro Thr Pro Val Leu Met Lys Ala 310 315 atc gtc atc catcag gcc aag gac ctg cgc gtc gag gac agc gcc gtc 1308 Ile Val Ile His GlnAla Lys Asp Leu Arg Val Glu Asp Ser Ala Val 320 325 330 gag gcg ccc ggcccc ggc gag gtg gag atc cgc ctt gcc gcc ggc ggc 1356 Glu Ala Pro Gly ProGly Glu Val Glu Ile Arg Leu Ala Ala Gly Gly 335 340 345 350 atc tgc ggctcg gac ctg cac tac tac aac cac ggc ggc ttc ggc acg 1404 Ile Cys Gly SerAsp Leu His Tyr Tyr Asn His Gly Gly Phe Gly Thr 355 360 365 gtg cgc ctcaag gag ccg atg atc ctc ggc cat gag gtt tcc ggc cac 1452 Val Arg Leu LysGlu Pro Met Ile Leu Gly His Glu Val Ser Gly His 370 375 380 gtc gcg gcgctc ggc gaa ggc gtc tcc ggc ctt gcc atc ggc gac ctc 1500 Val Ala Ala LeuGly Glu Gly Val Ser Gly Leu Ala Ile Gly Asp Leu 385 390 395 gtc gcc gtctcg ccc tcg cgg ccc tgc ggg gcg tgc gac tat tgc ctc 1548 Val Ala Val SerPro Ser Arg Pro Cys Gly Ala Cys Asp Tyr Cys Leu 400 405 410 aag ggc ttggcg aac cat tgc ttc aac atg cgc ttc tac ggc tcg gcc 1596 Lys Gly Leu AlaAsn His Cys Phe Asn Met Arg Phe Tyr Gly Ser Ala 415 420 425 430 atg cccttc ccg cac atc cag ggc gcg ttc cgc gag cgg ctg gtc gcc 1644 Met Pro PhePro His Ile Gln Gly Ala Phe Arg Glu Arg Leu Val Ala 435 440 445 aag gccagc cag tgc gtg aag gct gag ggc ctt tcg gca ggt gaa gcc 1692 Lys Ala SerGln Cys Val Lys Ala Glu Gly Leu Ser Ala Gly Glu Ala 450 455 460 gcg atggcc gag ccg ctc tcc gtc acg ctt cac gcc acg cgc cgg gcc 1740 Ala Met AlaGlu Pro Leu Ser Val Thr Leu His Ala Thr Arg Arg Ala 465 470 475 ggc gaaatg ctg ggc aag cgc gtg ctc gtc acc ggc tgc ggg ccg atc 1788 Gly Glu MetLeu Gly Lys Arg Val Leu Val Thr Gly Cys Gly Pro Ile 480 485 490 ggc accctg tcg atc ctc gcc gcc cgg cgc gcc ggc gcg gcg gag atc 1836 Gly Thr LeuSer Ile Leu Ala Ala Arg Arg Ala Gly Ala Ala Glu Ile 495 500 505 510 gtcgcc gct gac ctt tcc gag cgt gca ctc ggc ttt gcc cgc gcc gtc 1884 Val AlaAla Asp Leu Ser Glu Arg Ala Leu Gly Phe Ala Arg Ala Val 515 520 525 ggcgcg gac cgc acg gtc aac ctg tcg gaa gac cgc gac ggc ctc gtt 1932 Gly AlaAsp Arg Thr Val Asn Leu Ser Glu Asp Arg Asp Gly Leu Val 530 535 540 ccgttc agc gag aac aag gga tat ttc gat gtc ctc tac gaa tgc tcg 1980 Pro PheSer Glu Asn Lys Gly Tyr Phe Asp Val Leu Tyr Glu Cys Ser 545 550 555 ggcgcc cag ccg gcg ctg gtt gcc ggc atc cag gcc ttg cgc ccg cgc 2028 Gly AlaGln Pro Ala Leu Val Ala Gly Ile Gln Ala Leu Arg Pro Arg 560 565 570 ggcgtc atc gtc cag ctc ggc ctc ggc ggc gag atg agc ctt ccc atg 2076 Gly ValIle Val Gln Leu Gly Leu Gly Gly Glu Met Ser Leu Pro Met 575 580 585 590atg gcg atc acc gcc aag gaa ctg gac ctg cgc ggc tcc ttc cgc ttc 2124 MetAla Ile Thr Ala Lys Glu Leu Asp Leu Arg Gly Ser Phe Arg Phe 595 600 605cat gag gaa ttc gcc gtc gcc gtg aag ctg atg cag ggc ggc ctc atc 2172 HisGlu Glu Phe Ala Val Ala Val Lys Leu Met Gln Gly Gly Leu Ile 610 615 620gac gtg aag ccg ctg atc acc cat act ttg ccg ctt gcc gat gcg ctt 2220 AspVal Lys Pro Leu Ile Thr His Thr Leu Pro Leu Ala Asp Ala Leu 625 630 635cag gcc ttc gag atc gcc tcg gac aag ggg caa tcg atg aag act cag 2268 GlnAla Phe Glu Ile Ala Ser Asp Lys Gly Gln Ser Met Lys Thr Gln 640 645 650atc gca ttc agt taaggaagag cc atg agc atc cag ctt ttc gac ctc acg 2319Ile Ala Phe Ser Met Ser Ile Gln Leu Phe Asp Leu Thr 655 660 665 ggc aagcgc gcc ctc gtc acc ggc tcc tcg cag ggt atc ggc tat gcg 2367 Gly Lys ArgAla Leu Val Thr Gly Ser Ser Gln Gly Ile Gly Tyr Ala 670 675 680 ctc gccaag ggc ctt gcc gcc gcc ggc gcg gac atc gtc ctc aac ggc 2415 Leu Ala LysGly Leu Ala Ala Ala Gly Ala Asp Ile Val Leu Asn Gly 685 690 695 cgc gacgcg gcc aag ctg gcg gcc gcg gcg cag gaa ctc ggc gca aag 2463 Arg Asp AlaAla Lys Leu Ala Ala Ala Ala Gln Glu Leu Gly Ala Lys 700 705 710 715 cacacg ctc gcc ttc gac gcc acc gac cat gcc gcc gtg cgc gcg gcc 2511 His ThrLeu Ala Phe Asp Ala Thr Asp His Ala Ala Val Arg Ala Ala 720 725 730 atcgac gcc ttc gag gcg gag gtc ggc ccc atc gac atc ctc gtc aac 2559 Ile AspAla Phe Glu Ala Glu Val Gly Pro Ile Asp Ile Leu Val Asn 735 740 745 aatgcc ggc atg cag cac cgc acg ccg ctg gag gat ttc ccc gcc gat 2607 Asn AlaGly Met Gln His Arg Thr Pro Leu Glu Asp Phe Pro Ala Asp 750 755 760 gccttc gag cgc atc ctg aag acc aac atc tcg acg gtc ttc aat gtc 2655 Ala PheGlu Arg Ile Leu Lys Thr Asn Ile Ser Thr Val Phe Asn Val 765 770 775 ggccag gcc gtc gcg cgc cac atg atc gcg cgc ggc gcg ggc aag atc 2703 Gly GlnAla Val Ala Arg His Met Ile Ala Arg Gly Ala Gly Lys Ile 780 785 790 795atc aac atc gcc agc gtg cag acc gcg ctc gcc cgc ccc ggc atc gcg 2751 IleAsn Ile Ala Ser Val Gln Thr Ala Leu Ala Arg Pro Gly Ile Ala 800 805 810ccc tat acc gcc acc aag ggc gcc gtc ggc aac ctc acc aag ggc atg 2799 ProTyr Thr Ala Thr Lys Gly Ala Val Gly Asn Leu Thr Lys Gly Met 815 820 825gcg acc gac tgg gcg aaa tac ggc ctg caa tgc aac gcc atc gcg ccg 2847 AlaThr Asp Trp Ala Lys Tyr Gly Leu Gln Cys Asn Ala Ile Ala Pro 830 835 840ggc tat ttc gac acg ccg ctc aat gcc gcg ctg gtc gcc gat ccg gcc 2895 GlyTyr Phe Asp Thr Pro Leu Asn Ala Ala Leu Val Ala Asp Pro Ala 845 850 855ttt tcc gcc tgg ctg gaa aag cgc acg ccg gcc ggc cgc tgg ggc aag 2943 PheSer Ala Trp Leu Glu Lys Arg Thr Pro Ala Gly Arg Trp Gly Lys 860 865 870875 gtg gag gag ctg atc ggc gcc tgc atc ttt ctt tcc tcc gac gct tcc 2991Val Glu Glu Leu Ile Gly Ala Cys Ile Phe Leu Ser Ser Asp Ala Ser 880 885890 tcc ttc gtg aac gga cac acg ctc tat gtc gac ggc ggc atc acg gcc 3039Ser Phe Val Asn Gly His Thr Leu Tyr Val Asp Gly Gly Ile Thr Ala 895 900905 tcg ctc tgaggacaac aggcgcatcg tcctgatggg cgtcgccggc tgcggcaagt 3095Ser Leu ccgccgtcgg cgcggcgctc gccgcgcggc tcggtgcgat ccccgggtac cgagctcg3153 14 315 PRT Unknown environmental source 14 Met Ser Lys Ile Asp ValLeu Gln Val Gly Pro Tyr Pro Ala Trp Asp 1 5 10 15 Glu Glu Arg Leu AsnAla Thr Phe Thr Met His Arg Tyr Phe Glu Ala 20 25 30 Ala Asp Lys Ala AlaPhe Leu Ala Glu His Gly Gly Thr Ile Arg Gly 35 40 45 Ile Ala Thr Arg GlyGlu Leu Gly Ala Asn Arg Ala Met Ile Glu Ala 50 55 60 Leu Pro Lys Leu GluVal Ile Ser Val Tyr Gly Val Gly Phe Asp Ala 65 70 75 80 Val Asp Leu SerAla Ala Arg Glu Arg Gly Ile Arg Val Thr Asn Thr 85 90 95 Pro Asp Val LeuThr Lys Asp Val Ala Asp Leu Gly Ile Ala Met Met 100 105 110 Leu Ala GlnAla Arg Gly Val Ile Gly Gly Glu Ala Trp Val Lys Ser 115 120 125 Gly AspTrp Ala Ser Lys Gly Leu Tyr Pro Leu Lys Arg Arg Val His 130 135 140 GlyMet Arg Ala Gly Val Leu Gly Leu Gly Arg Ile Gly Tyr Glu Val 145 150 155160 Ala Lys Arg Leu Ala Gly Phe Asp Met Asp Ile Ala Tyr Ser Asp Thr 165170 175 Gly Pro Lys Asp Phe Ala Arg Asp Trp Thr Phe Val Ala Asp Pro Ala180 185 190 Glu Leu Ala Ala Arg Ser Asp Phe Leu Phe Val Thr Leu Ala AlaSer 195 200 205 Ala Glu Thr Arg His Ile Val Gly Arg Lys Val Ile Glu AlaLeu Gly 210 215 220 Pro Glu Gly Met Leu Ile Asn Ile Ser Arg Ala Ser AsnIle Asp Glu 225 230 235 240 Ser Ala Leu Leu Asp Ala Leu Glu Thr Lys AlaLeu Gly Ser Ala Ala 245 250 255 Leu Asp Val Phe Glu Gly Glu Pro Asn LeuAsn Pro Arg Phe Leu Ala 260 265 270 Leu Asp Asn Val Leu Leu Gln Pro HisMet Ala Ser Gly Thr Ile Glu 275 280 285 Thr Arg Lys Ala Met Gly Gln LeuVal Phe Asp Asn Leu Ser Ala His 290 295 300 Phe Asp Gly Arg Pro Leu ProThr Pro Val Leu 305 310 315 15 343 PRT Unknown environmental source 15Met Lys Ala Ile Val Ile His Gln Ala Lys Asp Leu Arg Val Glu Asp 1 5 1015 Ser Ala Val Glu Ala Pro Gly Pro Gly Glu Val Glu Ile Arg Leu Ala 20 2530 Ala Gly Gly Ile Cys Gly Ser Asp Leu His Tyr Tyr Asn His Gly Gly 35 4045 Phe Gly Thr Val Arg Leu Lys Glu Pro Met Ile Leu Gly His Glu Val 50 5560 Ser Gly His Val Ala Ala Leu Gly Glu Gly Val Ser Gly Leu Ala Ile 65 7075 80 Gly Asp Leu Val Ala Val Ser Pro Ser Arg Pro Cys Gly Ala Cys Asp 8590 95 Tyr Cys Leu Lys Gly Leu Ala Asn His Cys Phe Asn Met Arg Phe Tyr100 105 110 Gly Ser Ala Met Pro Phe Pro His Ile Gln Gly Ala Phe Arg GluArg 115 120 125 Leu Val Ala Lys Ala Ser Gln Cys Val Lys Ala Glu Gly LeuSer Ala 130 135 140 Gly Glu Ala Ala Met Ala Glu Pro Leu Ser Val Thr LeuHis Ala Thr 145 150 155 160 Arg Arg Ala Gly Glu Met Leu Gly Lys Arg ValLeu Val Thr Gly Cys 165 170 175 Gly Pro Ile Gly Thr Leu Ser Ile Leu AlaAla Arg Arg Ala Gly Ala 180 185 190 Ala Glu Ile Val Ala Ala Asp Leu SerGlu Arg Ala Leu Gly Phe Ala 195 200 205 Arg Ala Val Gly Ala Asp Arg ThrVal Asn Leu Ser Glu Asp Arg Asp 210 215 220 Gly Leu Val Pro Phe Ser GluAsn Lys Gly Tyr Phe Asp Val Leu Tyr 225 230 235 240 Glu Cys Ser Gly AlaGln Pro Ala Leu Val Ala Gly Ile Gln Ala Leu 245 250 255 Arg Pro Arg GlyVal Ile Val Gln Leu Gly Leu Gly Gly Glu Met Ser 260 265 270 Leu Pro MetMet Ala Ile Thr Ala Lys Glu Leu Asp Leu Arg Gly Ser 275 280 285 Phe ArgPhe His Glu Glu Phe Ala Val Ala Val Lys Leu Met Gln Gly 290 295 300 GlyLeu Ile Asp Val Lys Pro Leu Ile Thr His Thr Leu Pro Leu Ala 305 310 315320 Asp Ala Leu Gln Ala Phe Glu Ile Ala Ser Asp Lys Gly Gln Ser Met 325330 335 Lys Thr Gln Ile Ala Phe Ser 340 16 251 PRT Unknown environmentalsource 16 Met Ser Ile Gln Leu Phe Asp Leu Thr Gly Lys Arg Ala Leu ValThr 1 5 10 15 Gly Ser Ser Gln Gly Ile Gly Tyr Ala Leu Ala Lys Gly LeuAla Ala 20 25 30 Ala Gly Ala Asp Ile Val Leu Asn Gly Arg Asp Ala Ala LysLeu Ala 35 40 45 Ala Ala Ala Gln Glu Leu Gly Ala Lys His Thr Leu Ala PheAsp Ala 50 55 60 Thr Asp His Ala Ala Val Arg Ala Ala Ile Asp Ala Phe GluAla Glu 65 70 75 80 Val Gly Pro Ile Asp Ile Leu Val Asn Asn Ala Gly MetGln His Arg 85 90 95 Thr Pro Leu Glu Asp Phe Pro Ala Asp Ala Phe Glu ArgIle Leu Lys 100 105 110 Thr Asn Ile Ser Thr Val Phe Asn Val Gly Gln AlaVal Ala Arg His 115 120 125 Met Ile Ala Arg Gly Ala Gly Lys Ile Ile AsnIle Ala Ser Val Gln 130 135 140 Thr Ala Leu Ala Arg Pro Gly Ile Ala ProTyr Thr Ala Thr Lys Gly 145 150 155 160 Ala Val Gly Asn Leu Thr Lys GlyMet Ala Thr Asp Trp Ala Lys Tyr 165 170 175 Gly Leu Gln Cys Asn Ala IleAla Pro Gly Tyr Phe Asp Thr Pro Leu 180 185 190 Asn Ala Ala Leu Val AlaAsp Pro Ala Phe Ser Ala Trp Leu Glu Lys 195 200 205 Arg Thr Pro Ala GlyArg Trp Gly Lys Val Glu Glu Leu Ile Gly Ala 210 215 220 Cys Ile Phe LeuSer Ser Asp Ala Ser Ser Phe Val Asn Gly His Thr 225 230 235 240 Leu TyrVal Asp Gly Gly Ile Thr Ala Ser Leu 245 250 17 32 DNA ArtificialSequence synthetic construct 17 acccaagctt caccaaaaga gtgaagagga ag 3218 34 DNA Artificial Sequence synthetic construct 18 cgtatctagaaaaatattct ggtgatgaag gtga 34 19 34 DNA Artificial Sequence syntheticconstruct 19 agactctaga tccacataaa cgcactgcgt aaac 34 20 33 DNAArtificial Sequence synthetic construct 20 gaggggatcc tggcttcgtgaacgatatac tgg 33 21 31 DNA Artificial Sequence synthetic construct 21aataggatcc ttcatcacca gaatattttt a 31 22 28 DNA Artificial Sequencesynthetic construct 22 cataggtacc ggctttcaga taggtgcc 28

What is claimed is:
 1. An isolated nucleic acid molecule encoding apolypeptide which has 2,5-DKG permease activity.
 2. The isolated nucleicacid molecule of claim 1, comprising a nucleotide sequence having atleast 40% identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7, 9 and
 11. 3. The isolated nucleicacid molecule of claim 1, comprising a nucleotide sequence having atleast 80% identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7, 9 and
 11. 4. The isolated nucleicacid molecule of claim 1, comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NOS: 1, 3, 5, 7, 9 and
 11. 5. Theisolated nucleic acid molecule of claim 1, comprising a nucleotidesequence which encodes a polypeptide having at least 40% identity to anamino acid sequence selected from the group consisting of SEQ ID NOS: 2,4, 6, 8, 10 and
 12. 6. The isolated nucleic acid molecule of claim 1,comprising a nucleotide sequence which encodes a polypeptide having atleast 80% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10 and
 12. 7. The isolated nucleicacid molecule of claim 1, which encodes a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6,8, 10 and
 12. 8. The isolated nucleic acid molecule of claim 1, whichcomprises a nucleotide sequence encoding a peptide having at least 10contiguous amino acids of any of SEQ ID NOS: 2, 4, 6, 8, 10 and
 12. 9.The isolated nucleic acid molecule of claim 1, which comprises anucleotide sequence encoding a peptide having at least 10 contiguousamino acids of at least any two of SEQ ID NOS: 4, 8 and
 12. 10. Theisolated nucleic acid molecule of claim 1, which comprises a nucleotidesequence encoding a peptide having at least 10 contiguous amino acids ofat least any two of SEQ ID NOS: 2, 6 and
 10. 11. The isolated nucleicacid molecule of claim 1 operatively linked to a promoter of geneexpression.
 12. The isolated nucleic acid molecule of claim 11, whereinsaid promoter is a lac promoter.
 13. A vector comprising the isolatednucleic acid molecule of claim
 11. 14. The vector of claim 13,comprising a spectinomycin resistance gene.
 15. A bacterial cell,comprising the vector of claim
 13. 16. The bacterial cell of claim 15,wherein said isolated nucleic acid molecule comprises a nucleotidesequence which encodes a polypeptide having an amino acid sequence atleast 80% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 8, 10 and
 12. 17. The bacterial cellof claim 16, wherein said amino acid sequence is at least 95% identicalto SEQ ID NO:
 8. 18. The bacterial cell of claim 17, further comprisingan isolated nucleic acid molecule comprising a nucleotide sequence whichencodes a polypeptide having an amino acid sequence at least 95%identical to SEQ ID NO:
 4. 19. The bacterial cell of claim 17, furthercomprising an isolated nucleic acid molecule comprising a nucleotidesequence which encodes a polypeptide having an amino acid sequence atleast 95% identical to SEQ ID NO:
 10. 20. The bacterial cell of claim15, which is of the genus Klebsiella.
 21. The bacterial cell of claim15, which is deficient in endogenous 2,5-DKG activity.
 22. The bacterialcell of claim 21, comprising an isolated nucleic acid molecule encodinga polypeptide having at least 80% identity to SEQ ID NO: 14 and 2-ketoreductase activity.
 23. The bacterial cell of claim 21, comprising anisolated nucleic acid molecule encoding a polypeptide having at least80% identity to SEQ ID NO: 16 and 5-keto reductase activity.
 24. Thebacterial cell of claim 15, which is of the genus Pantoea.
 25. Thebacterial cell of claim 15, which expresses an enzyme that catalyzes theconversion of 2,5-DKG to 2-KLG.
 26. The bacterial cell of claim 25,which expresses enzymes that catalyze the conversion of glucose to2,5-DKG.
 27. The bacterial cell of claim 26, which is deficient inendogenase 2-keto-reductase activity.
 28. An isolated oligonucleotide,comprising at least 20 contiguous nucleotides of a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 1, 3, 5, 7 and
 9. 29.The isolated oligonucleotide of claim 28, comprising at least 50contiguous nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7 and
 9. 30. An isolatedoligonucleotide, comprising a nucleotide sequence encoding a peptidehaving at least 10 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2, 4, 6, 8 and
 10. 31.A method of making the isolated nucleic acid molecule of claim 1,comprising introducing into a bacterial cell deficient in endogenous2,5-DKG permease activity one or more expressible nucleic acidmolecules, identifying a cell having 2,5-DKG permease activity followingsaid introduction, and isolating the introduced nucleic acid moleculefrom said cell.
 32. The method of claim 31, wherein said one or moreisolated nucleic acid molecules is a genomic DNA library.
 33. The methodof claim 32, wherein said genomic DNA library is prepared from anenvironmental sample.
 34. The method of claim 31, wherein said bacterialcell is a Klebsiella oxytoca deficient in yiaX2.
 35. The method of claim31, wherein said bacterial cell comprises an isolated nucleic acidmolecule encoding a polypeptide having at least 80% identity to SEQ IDNO: 14 and 2-keto reductase activity, and a polypeptide having at least80% identity to SEQ ID NO: 16 and 5-keto reductase activity.
 36. Amethod of using the isolated nucleic acid molecule of claim 1 to enhance2-KLG production, comprising expressing the polypeptide encoded by saidnucleic acid molecule in a bacterial which expresses an enzyme thatcatalyzes the conversion of 2,5-DKG to 2-KLG.
 37. The method of claim36, wherein said bacterial cell further expresses enzymes that catalyzethe conversion of glucose to 2,5-DKG.
 38. The method of claim 37,wherein said bacterial cell is deficient in endogenase 2-keto reductaseactivity.
 39. The method of claim 36, wherein said bacterial cell is ofthe genus Pantoea.
 40. The method of claim 36, further comprisingconverting said 2-KLG to ascorbic acid.
 41. An isolated polypeptidewhich has 2,5-DKG permease activity.
 42. The isolated polypeptide ofclaim 41, comprising an amino acid sequence having at least 40% identityto an amino acid sequence selected from the group consisting of SEQ IDNOS: 2, 4, 6, 8, 10 and
 12. 43. The isolated polypeptide of claim 41,comprising an amino acid sequence having at least 80% identity to anamino acid sequence selected from the group consisting of SEQ ID NOS: 2,4, 6, 8, 10 and
 12. 44. The isolated polypeptide of claim 41, comprisingan amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10 and
 12. 45. The isolated polypeptide of claim 41,comprising at least 10 contiguous amino acids of any of SEQ ID NOS: 2,4, 6, 8, 10 and
 12. 46. An isolated peptide, comprising at least 10contiguous amino acids of any of SEQ ID NOS: 2, 4, 6, 8 and 10, whereinsaid peptide is immunogenic.
 47. An antibody specific for the isolatedpolypeptide of claim
 44. 48. An antibody specific for the isolatedpeptide of claim 46.